Modified Mini-Hepcidin Peptides and Methods of Using Thereof

ABSTRACT

Disclosed herein are peptides which exhibit hepcidin activity and methods of making and using thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Ser. No.61/568,724, filed 9 Dec. 2011, which is herein incorporated by referencein its entirety.

This application is related to U.S. patent application Ser. No.13/131,792, which is a 371 National Phase entry of PCT/US2009/066711,filed 4 Dec. 2009, and U.S. Provisional Application Ser. No. 61/120,277,filed 5 Dec. 2008, all of which are herein incorporated by reference intheir entirety.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.NIH/NIDDK R01 DK090554, awarded by the National Institutes of Health.The Government has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“034044_097WO1_ST25” which is 2.53 kb in size was created on 1 Nov. 2012and electronically submitted via EFS-Web herewith the application isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to peptides which exhibithepcidin activity.

2. Description of the Related Art

Hepcidin, a peptide hormone produced by the liver, is a regulator ofiron homeostasis in humans and other mammals. Hepcidin acts by bindingto its receptor, the iron export channel ferroportin, and causing itsinternalization and degradation. Human hepcidin is a 25-amino acidpeptide (Hep25). See Krause et al. (2000) FEBS Lett 480:147-150, andPark et al. (2001) J Biol Chem 276:7806-7810. The structure of thebioactive 25-amino acid form of hepcidin is a simple hairpin with 8cysteines that form 4 disulfide bonds as described by Jordan et al.(2009) J Biol Chem 284:24155-67. The N terminal region is required foriron-regulatory function, and deletion of 5 N-terminal amino acidresidues results in a loss of iron-regulatory function. See Nemeth etal. (2006) Blood 107:328-33.

Abnormal hepcidin activity is associated with iron overload diseaseswhich include hereditary hemochromatosis and iron-loading anemias andmyelodysplasia. Hereditary hemochromatosis (HH) is a genetic ironoverload disease that is mainly caused by hepcidin deficiency, or veryrarely by hepcidin resistance. This allows excessive absorption of ironfrom the diet and development of iron overload. Clinical manifestationsof HH may include liver disease (hepatic cirrhosis, hepatocellularcarcinoma), diabetes, and heart failure. Currently, the only treatmentfor HH is regular phlebotomy, which is effective but very burdensome forthe patients.

Iron-loading anemias are hereditary anemias with ineffectiveerythropoiesis such as β-thalassemia, which are accompanied by severeiron overload. Complications from iron overload are the main cause ofmorbidity and mortality for these patients. Hepcidin deficiency is themain cause of iron overload in untransfused patients, and contributes toiron overload in transfused patients. The current treatment for ironoverload in these patients is iron chelation which is very burdensome,sometimes ineffective and accompanied by frequent side effects.

SUMMARY OF THE INVENTION

The present invention generally relates to peptides which exhibithepcidin activity and methods of using thereof.

The present invention provides peptides, which may be isolated and/orpurified, comprising, consisting essentially or consisting of thefollowing Structural Formula IA or IB:

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10  IA

A10-A9-A8-A7-A6-A5-A4-A3-A2-A1  IB

wherein

-   A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D-pyroglutamate, Gln,    D-Gln, Asn, D-Asn, or an unnatural amino acid commonly used as a    substitute thereof such as bhAsp, Ida, Ida(NHPal), and N-MeAsp,    preferably Ida and N-MeAsp;-   A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile, Ala, D-Ala or    an unnatural amino acid commonly used as a substitute thereof such    as Tle, Inp, Chg, bhThr, and N-MeThr;-   A3 is His, D-His, Asn, D-Asn, Arg, D-Arg, or an unnatural amino acid    commonly used as a substitute thereof such as L-His(π-Me),    D-His(π-Me), L-His(τ-Me), or D-His(τ-Me);-   A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr, D-Tyr, or    an unnatural amino acid commonly used as a substitute thereof such    as Phg, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, Amc, PheF5, hPhe, Igl,    or cyclohexylalanine, preferably Dpa;-   A5 is Pro, D-Pro, Ser, D-Ser, or an unnatural amino acid commonly    used as a substitute thereof such as Oic, bhPro, trans-4-PhPro,    cis-4-PhPro, cis-5-PhPro, and Idc, preferably bhPro;-   A6 is Arg, D-Arg, Ile, D-Ile, Leu, D-Leu, Thr, D-Thr, Lys, D-Lys,    Val, D-Val, or an unnatural amino acid commonly used as a substitute    thereof such as D-Nω,ω-dimethyl-arginine, L-Nω,ω-dimethyl-arginine,    D-homoarginine, L-homoarginine, D-norarginine, L-norarginine,    citrulline, a modified Arg wherein the guanidinium group is modified    or substituted, Norleucine, norvaline, bhIle, Ach, N-MeArg, and    N-MeIle, preferably Arg;-   A7 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, or an unnatural amino acid    such as Cys(S-tBut), homoCys, Pen, (D)Pen, preferably S-tertiary    butyl-cysteine, Cys(S-S-Pal), Cys(S-S-cysteamine-Pal),    Cys(S-S-Cys-NHPal), and Cys(S-S-Cys);-   A8 is Arg, D-Arg, Ile, D-Ile, Leu, D-Leu, Thr, D-Thr, Lys, D-Lys,    Val, D-Val, or an unnatural amino acid commonly used as a substitute    thereof such as D-Nω,ω-dimethyl-arginine, L-Nω,ω-dimethyl-arginine,    D-homoarginine, L-homoarginine, D-norarginine, L-norarginine,    citrulline, a modified Arg wherein the guanidinium group is modified    or substituted, Norleucine, norvaline, bhIle, Ach, N-MeArg, and    N-MeIle, preferably Arg;-   A9 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Tyr, D-Tyr, Trp, D-Trp,    Phe-R^(a), D-Phe-R^(a), Dpa-R^(a), D-Dpa-R^(a), Trp-R^(a),    bhPhe-R^(a), or an unnatural amino acid commonly used as a    substitute thereof such as PheF5, N-MePhe, benzylamide,    2-aminoindane, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, and    cyclohexylalanine, which may or may not have R^(a) linked thereto,    preferably bhPhe and bhPhe-R^(a), wherein R^(a) is palmitoyl-PEG-,    wherein PEG is PEG11 or miniPEG3, palmitoyl-PEG-PEG, wherein PEG is    PEG11 or miniPEG3, butanoyl (C4)-PEG11-, octanoyl (C8,    Caprylic)-PEG11-, palmitoyl (C16)-PEG11-, or tetracosanoyl (C24,    Lignoceric)-PEG11-; and-   A10 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, or an unnatural amino    acid such as Ida, Ida(NHPal)Ahx, and Ida(NBzl2)Ahx;    wherein the carboxy-terminal amino acid is in amide or carboxy-form;    wherein at least one sulfhydryl amino acid is present as one of the    amino acids in the sequence; and wherein A1, A1 to A2, A10, or a    combination thereof are optionally absent, with the proviso that the    peptide is not one of the peptides as set forth in Table 1. In some    embodiments, the peptides of the present invention contain at least    one of the following: a) A1=N-MeAsp, Ida, or Ida(NHPal); b)    A5=bhPro; c) A6=D-Val, D-Leu, Lys, D-Lys, Arg, D-Arg, Ach, bhArg, or    N-MeArg; d) A7=Cys(S-S-Pal), Cys(S-S-cysteamine-Pal),    Cys(S-S-Cys-NHPal), or Cys(S-S-Cys); and/or e) A8=D-Val, D-Leu, Lys,    D-Lys, Arg, D-Arg, Ach, bhArg, or N-MeArg. In some embodiments, i)    when A1 is Ida and A9 is Phe, then A10 is not Ahx-Ida(NHPal); ii)    when A1 is Ida, A9 is not bhPhe-R^(b), wherein R^(b) is    S-(palmityl)thioglycolic-PEG-; iii) when A4 is D-Phe, A7 is not    D-Cys(S-S-tBut) and A9 is not D-Trp-R^(c), wherein R^(c) is    Butanoyl-PEG11-, Octanoyl-PEG11-, Palmitoyl-PEG11-, or    Tetracosanoyl-PEG11-; or iv) when A1 is Ida and A9 is bhPhe-R^(d),    wherein R^(d) is palmitoyl-PEG-miniPEG3-, A6 and A8 are not both    D-Arg or both bhArg. In some embodiments, A1 is D-Asp, N-MeAsp, Ida,    or Ida(NHPal); A2 is Thr or D-Thr; A3 is His or D-His; A4 is Dpa or    D-Dpa; A5 is Pro, D-Pro, bhPro, or Oic; A6 is Ile, D-Ile, Arg,    D-Val, D-Leu, Ach, or N-MeArg; A7 is Cys, D-Cys, Cys(S-S-Pal),    Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys); A8 is    Ile, D-Ile, Arg, D-Val, D-Leu, Ach, or N-MeArg; A9 is Phe, D-Phe,    Dpa, D-Dpa, Trp, D-Trp, bhPhe, Phe-R^(a), D-Phe-R^(a), Dpa-R^(a),    D-Dpa-R^(a), Trp-R^(a), bhPhe-R^(a), wherein R^(a) is    palmitoyl-PEG-, wherein PEG is PEG11 or miniPEG3, palmitoyl-PEG-PEG,    wherein PEG is PEG11 or miniPEG3, butanoyl (C4)-PEG11-, octanoyl    (C8, Caprylic)-PEG11-, palmitoyl (C16)-PEG11-, or tetracosanoyl    (C24, Lignoceric)-PEG11-; and A10, if present, is Ida(NHPal)Ahx or    Ida(NBzl2)Ahx. In some embodiments, A6 and/or A8 is a lysine    derivative such as N-ε-Dinitrophenyl-lysine, N-ε-Methyl-lysine,    N,N-ε-Dimethyl-lysine, and N,N,N-ε-Trimethyl-lysine. In some    embodiments, the peptide is selected from the group consisting of:    PR42′, PR47, PR48, PR49, PR50, PR51, PR52, PR53, PR56, PR57, PR58,    PR59, PR60, PR61, PR63, PR65, PR66, PR67, PR68, PR69, PR70, PR71,    PR72, PR73, PR74, and PR82.

In some embodiments, the peptides form a cyclic structure by a disulfidebond. In some embodiments, the peptides exhibit hepcidin activity. Insome embodiments, the peptides bind ferroportin, preferably humanferroportin.

In some embodiments, the present invention provides compositions andmedicaments which comprise at least one peptide, which may be isolated,synthesized and/or purified, comprising, consisting essentially orconsisting of Structural Formula IA or IB as set forth herein. In someembodiments, the present invention provides method of manufacturingmedicaments for the treatment of diseases of iron metabolism, such asiron overload diseases, which comprise at least one peptide, which maybe isolated and/or purified, comprising, consisting essentially orconsisting of Structural Formula IA or IB as set forth herein. Alsoprovided are methods of treating a disease of iron metabolism in asubject, such as a mammalian subject, preferably a human subject, whichcomprises administering at least one peptide, which may be isolatedand/or purified, comprising, consisting essentially or consisting ofStructural Formula IA or IB as set forth herein or a compositioncomprising said at least one peptide to the subject. In someembodiments, the peptide is administered in a therapeutically effectiveamount. In some embodiments, the therapeutically effective amount is aneffective daily dose administered as a single daily dose or as divideddaily doses. The peptides of the present invention can also beadministered at a variety of doses.

In some embodiments the dose is given as a weekly dose, e.g. from1-10,000 μg/kg/dose. In some embodiments, the daily dose is about1-1,000, preferably about 10-500 μm/kg/day. Dosages can vary accordingto the type of formulation of peptidyl drug administered as well as theroute of administration. One skilled in the art can adjust the dosage bychanging the route of administration or formulation, so that the dosageadministered would result in a similar pharmacokinetic or biologicalprofile as would result from the preferred dosage ranges describedherein. In some embodiments, the composition to be administered isformulated for oral, pulmonary or mucosal administration.

Some embodiments include any dosage with any route of administrationwhich results in an effective pharmacokinetic and pharmacodynamicprofile by reducing serum iron values by 10-80%. Some preferred dosesinclude those that result in a desired reduction in serum iron.Administration of the peptidyl or protein formulations of the presentinvention includes both direct administration, includingself-administration, and indirect administration, including the act ofprescribing a drug. For example, a physician who instructs a patient toself-administer a drug and/or provides a patient with a prescription fora drug is considered to be administering the drug to the patient.

In some embodiments, the present invention provides methods of binding aferroportin or inducing ferroportin internalization and degradationwhich comprises contacting the ferroportin with at least one peptide orcomposition as disclosed herein.

In some embodiments, the present invention provides kits comprising atleast one peptide or composition as disclosed herein packaged togetherwith a reagent, a device, instructional material, or a combinationthereof.

In some embodiments, the present invention provides complexes whichcomprise at least one peptide as disclosed herein bound to aferroportin, preferably a human ferroportin, or an antibody, such as anantibody which specifically binds a peptide as disclosed herein, Hep25,or a combination thereof.

In some embodiments, the present invention provides the use of at leastone peptide, which may be isolated and/or purified, comprising,consisting essentially or consisting of Structural Formula IA or IB asset forth herein or a composition comprising, consisting essentially of,or consisting of said at least one peptide for the manufacture of amedicament for treating a disease of iron metabolism and/or lowering theamount of iron in a subject in need thereof, wherein the medicament isprepared to be administered at an effective daily dose, as a singledaily dose, or as divided daily doses. In some embodiments, the dose isabout 1-1,000, preferably about 10-500 μm/kg/day. In some embodiments,the medicament is formulated for subcutaneous injection or oral,pulmonary or mucosal administration.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are intended toprovide further explanation of the invention as claimed. Theaccompanying drawings are included to provide a further understanding ofthe invention and are incorporated in and constitute part of thisspecification, illustrate several embodiments of the invention, andtogether with the description serve to explain the principles of theinvention.

DESCRIPTION OF THE DRAWINGS

This invention is further understood by reference to the drawingswherein:

FIG. 1 is a graph showing the relative hepcidin activity of alaninesubstitutions in Hep25.

FIG. 2A is a graph showing the relative hepcidin activities of F4substitutions in Hep25.

FIG. 2B is a graph showing the relative hepcidin activities of F9substitutions in Hep25.

FIG. 3A is a graph showing the hepcidin activities of Hep1-9 and Hep1-10C7A relative to Hep25 (A).

FIG. 3B is a graph showing the hepcidin activities of Hep1-7 and Hep1-8relative to Hep1-9 or Hep25.

FIG. 3C is a graph showing the hepcidin activities of Hep4-7, Hep3-7,Hep3-8 and Hep3-9 relative to Hep25.

FIG. 4 is a graph showing the hepcidin activities of C7 modifiedpeptides relative to Hep25 and Hep 1-9.

FIG. 5 is a graph showing in vivo effect (as measured by serum ironlevels in mice) of mini-hepcidins Hep1-9, PR6 and PR12 compared to Hep25or control (PBS). The peptides were injected intraperitoneally, 50 μgpeptide per mouse.

FIG. 6 is a graph showing in vivo effect (as measured by serum ironlevels in mice) of mini-hepcidin PR27 injected intraperitoneally (20 and200 nmoles). The amount of injected Hep25 was 20 nmoles.

FIG. 7 is a graph showing in vivo effect (as measured by serum ironlevels in mice) of mini-hepcidin riHep7ΔDT injected intraperitoneally(20 and 200 nmoles). The amount of injected Hep25 was 20 nmoles.

FIG. 8 is a graph showing in vivo effect (as measured by serum ironlevels in mice) of mini-hepcidins PR27 and PR28 which were first mixedwith liposomes and injected intraperitoneally (20 nmoles). The amount ofinjected Hep25 was 20 nmoles.

FIG. 9 is a graph showing in vivo effect (as measured by serum ironlevels in mice) of mini-hepcidin PR27 after oral administration bygavage (200 nmoles).

FIGS. 10A-10C show mini-hepcidin PR65 and its activity in wild-typemice. FIG. 10A shows the structural formula of PR65. Ida=iminodiaceticacid, Dpa=diphenylalanine, bhPro=beta-homo proline, bhPhe=beta-homophenylalanine. FIG. 10B shows the serum iron in wild-type C57BL/6 mice 4hours after intraperitoneal injection of solvent, native hepcidin orPR65 (n=4-8 in each group). **p=0.01, *p=0.005. FIG. 10C shows the serumiron in wild-type C57BL/6 mice 4 hours after intraperitoneal orsubcutaneous injection of 20 nmoles of PR65 (n=4 in each group).*p=0.007, **p=0.04. In FIGS. 10B and 10C the bars represent mean valuesand error bars standard deviations.

FIGS. 11A and 11B show the hypoferremic effect of PR65 in iron-loadedhepcidin knockout mice. FIG. 11A shows that PR65 induced adose-dependent decrease in serum iron 24 hours after a subcutaneousinjection. Mean values and standard deviations are shown, n=3-5 mice perpoint. #p=0.005, &p=0.004, *p<0.001. FIG. 11B shows the time course ofhypoferremia induced by a subcutaneous injection of 100 nmoles of PR65.Mean and standard deviations are shown, n=4-6 mice per point. #p=0.008,*p<0.001.

FIG. 12 shows the changes in iron distribution in PR65-treated hepcidinknockout mice. Tissue iron was visualized by enhanced Perls stain at0-48 hours after subcutaneous injection of PR65 (100 nmoles).Representative images are shown. Horizontal bars indicate 400 μm (10×)and 100 μm (40×). Top row: Spleen iron was scant and its distributiondid not change appreciably during the 48 hours. Middle row: Iron in thevillus stroma was evident in solvent-treated and 1-4 hour PR65-treatedmice, indicating active ferroportin-mediated efflux of iron frombasolateral membranes of enterocytes. From 12-24 hours, iron wasretained in enterocytes consistent with (mini)hepcidin-inducedferroportin degradation. 48 hours after injection iron was no longerretained by enterocytes. Bottom row: As expected, the livers wereiron-loaded at baseline and no changes in the pattern of iron stainingwere seen within 48 hours of PR65 treatment.

FIGS. 13A-13E show that PR65 prevented iron loading in iron-depletedhepcidin knockout mice. All mice were placed on an iron-deficient diet(4 ppm iron) from ages 5-6 weeks until 12 weeks. The “baseline” group(n=7) was examined at 12 weeks of age (white bars). The rest of the micewere fed an iron-loading diet (300 ppm) for 2 more weeks while receivingdaily subcutaneous injections of solvent (grey bars, n=6) or PR65 at 20,50 or 100 nmoles per day (black bars, n=4 per dose). The mice wereanalyzed 24 hours after the last injection. Compared to solvent, PR65injections resulted in: FIG. 13A—iron retention in the spleen; FIG.13B—a dose-dependent decrease in serum iron; FIG. 13C—a correspondingdose-dependent decrease in Hb levels; FIG. 13D—a decrease in heart ironat higher doses; and FIG. 13E—decreased liver iron. Liver iron contentin PR65-injected mice did not significantly differ from that in thebaseline group of mice, indicating that little to no new iron wasabsorbed or deposited in the liver during the 2-week treatment. Graphsshow means and standard deviations. Student's t-test was used to comparethe mean of each condition to that of solvent treatment (p value overbars). In FIG. 13E, mean of each condition was also compared to thebaseline (p values at lines over bars).

FIG. 14 shows the cellular distribution of iron after 2 weeks of PR65injections for the prevention of iron overload. Representative imagesare shown. Horizontal bars indicate 400 μm (10×) and 100 μm (40×). Ironaccumulation was seen in the splenic red pulp of PR65-treated mice butnot solvent-treated mice. Similarly, iron accumulation in duodenalenterocytes was seen only in PR65-treated mice. Compared to heart ironstaining of solvent-injected mice, there was less iron accumulation inthe heart of animals injected with 50 and 100 nmoles of PR65, consistentwith the quantitative method in FIG. 4. Liver iron loading in micetreated with 20 and 50 nmoles of PR65 was similar to that of thebaseline group and much less than the iron loading in thesolvent-treated group. At the highest PR65 dose, liver iron was lowerthan at baseline indicating that mice were able to mobilize liver irondespite high mini-hepcidin activity.

FIGS. 15A-15E shows that two-week PR65 treatment of iron-loaded hepcidinknockout mice caused modest redistribution of iron. Hepcidin knockoutmice were kept on a 300 ppm iron diet for their entire lifespan.Starting at 12 weeks of age, one group of mice was injectedsubcutaneously with solvent (n=4) and the other with 50 nmoles of PR65(n=4) daily for 2 weeks. Iron and hematological parameters were measured24 hours after the last injection. In PR65-treated mice compared tosolvent-treated mice: FIG. 15A—spleen iron increased more than 15-foldconfirming PR65 activity; FIG. 15B—serum iron concentrations weresimilar 24 hours after the last injection; FIG. 15C—hemoglobin decreasedby 2 g/dL indicating iron restriction to erythropoiesis; FIG. 15D—heartiron tended to decrease, though the difference was not statisticallysignificant at the number of mice tested; FIG. 15E—liver iron decreasedby about 20%.

FIG. 16 shows the cellular distribution of iron after 2 weeks of PR65injections for the treatment of established iron overload. Tissuesections correspond to the animals analyzed in FIGS. 15A-15E, withrepresentative images shown. Horizontal bars indicate 400 μm (10×) and100 μm (40×). Enhanced Perls stain confirmed that splenic macrophagesand duodenal enterocytes retained iron in PR65-treated but not insolvent-treated mice. Compared to solvent-treated controls, less intenseiron staining was observed in the liver of mice treated with PR65. Noconsistent differences between solvent- and PR65-treated mice were seenin sections of the heart (not shown).

FIG. 17 shows some of the structures of the molecules recited in Tables1, 3 and 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptides which are useful in the studyand treatment of diseases of iron metabolism.

As used herein, a “disease of iron metabolism” includes diseases whereaberrant iron metabolism directly causes the disease, or where ironblood levels are dysregulated causing disease, or where irondysregulation is a consequence of another disease, or where diseases canbe treated by modulating iron levels, and the like. More specifically, adisease of iron metabolism according to this disclosure includes ironoverload diseases, iron deficiency disorders, disorders of ironbiodistribution, other disorders of iron metabolism and other disorderspotentially related to iron metabolism, etc. Diseases of iron metabolisminclude hemochromatosis, HFE mutation hemochromatosis, ferroportinmutation hemochromatosis, transferrin receptor 2 mutationhemochromatosis, hemojuvelin mutation hemochromatosis, hepcidin mutationhemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis,hepcidin deficiency, transfusional iron overload, thalassemia,thalassemia intermedia, alpha thalassemia, sideroblastic anemia,porphyria, porphyria cutanea tarda, African iron overload,hyperferritinemia, ceruloplasmin deficiency, atransferrinemia,congenital dyserythropoietic anemia, anemia of chronic disease, anemiaof inflammation, anemia of infection, hypochromic microcytic anemia,iron-deficiency anemia, iron-refractory iron deficiency anemia, anemiaof chronic kidney disease, erythropoietin resistance, iron deficiency ofobesity, other anemias, benign or malignant tumors that overproducehepcidin or induce its overproduction, conditions with hepcidin excess,Friedreich ataxia, gracile syndrome, Hallervorden-Spatz disease,Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma,cancer, hepatitis, cirrhosis of liver, pica, chronic renal failure,insulin resistance, diabetes, atherosclerosis, neurodegenerativedisorders, multiple sclerosis, Parkinson's disease, Huntington'sdisease, and Alzheimer's disease. As used herein, “iron overloaddiseases” and “diseases of iron overload” refer diseases and disordersthat result in or may cause abnormally high levels of iron in afflictedsubjects if untreated.

In some cases the diseases and disorders included in the definition of“disease of iron metabolism” are not typically identified as being ironrelated. For example, hepcidin is highly expressed in the murinepancreas suggesting that diabetes (Type I or Type II), insulinresistance, glucose intolerance and other disorders may be amelioratedby treating underlying iron metabolism disorders. See Ilyin, G. et al.(2003) FEBS Lett. 542 22-26, which is herein incorporated by reference.As such, these diseases are encompassed under the broad definition.Those skilled in the art are readily able to determine whether a givendisease is a “disease or iron metabolism” according to the presentinvention using methods known in the art, including the assays of WO2004092405, which is herein incorporated by reference, and assays whichmonitor hepcidin, hemojuvelin, or iron levels and expression, which areknown in the art such as those described in U.S. Pat. No. 7,534,764,which is herein incorporated by reference.

In preferred embodiments of the present invention, the diseases of ironmetabolism are iron overload diseases, which include hereditaryhemochromatosis, iron-loading anemias, alcoholic liver diseases andchronic hepatitis C.

As used herein, the terms “protein”, “polypeptide” and “peptide” areused interchangeably to refer to two or more amino acids linkedtogether. Except for the abbreviations for the uncommon or unnaturalamino acids set forth in Table 2 below, the three-letter and one-letterabbreviations, as used in the art, are used herein to represent aminoacid residues. Except when preceded with “D-”, the amino acid is anL-amino acid. Groups or strings of amino acid abbreviations are used torepresent peptides. Except when specifically indicated, peptides areindicated with the N-terminus on the left and the sequence is writtenfrom the N-terminus to the C-terminus.

The peptides of the present invention may be made using methods known inthe art including chemical synthesis (solid-phase, solution phase, or acombination of both), biosynthesis or in vitro synthesis usingrecombinant DNA methods. See e.g. Kelly & Winkler (1990) GeneticEngineering Principles and Methods, vol. 12, J. K. Setlow ed., PlenumPress, NY, pp. 1-19; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten(1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid PhasePeptide Synthesis, 2ed. Pierce, Rockford, Ill., which are hereinincorporated by reference. The peptides of the present invention may bepurified using protein purification techniques known in the art such asreverse phase high-performance liquid chromatography (HPLC),ion-exchange or immunoaffinity chromatography, precipitation,filtration, size exclusion, or electrophoresis. See Olsnes, S. and A.Pihl (1973) Biochem. 12(16):3121-3126; and Scopes (1982) ProteinPurification, Springer-Verlag, NY, which are herein incorporated byreference. Alternatively, the peptides of the present invention may bemade by recombinant DNA techniques known in the art. Thus,polynucleotides that encode the polypeptides of the present inventionare contemplated herein. In preferred embodiments, the polynucleotidesare isolated. As used herein “isolated polynucleotides” refers topolynucleotides that are in an environment different from that in whichthe polynucleotide naturally occurs.

In some embodiments, the peptides of the present invention aresubstantially purified. As used herein, a “substantially purified”compound refers to a compound that is removed from its naturalenvironment and is at least about 60% free, preferably about 75% free,and most preferably about 90% free from other macromolecular componentswith which the compound is naturally associated, or a compound that isat least about 60% free, preferably about 75% free, and most preferablyabout 90% free from other peptide components as measured by HPLC withdetection at 214 nm.

As used herein, an “isolated” compound refers to a compound which isisolated from its native environment. For example, an isolated peptideis one which does not have its native amino acids, which correspond tothe full length polypeptide, flanking the N-terminus, C-terminus, orboth. For example, isolated Hep1-9 refers to an isolated peptidecomprising amino acid residues 1-9 of Hep25 which may have non-nativeamino acids at its N-terminus, C-terminus, or both, but does not have acysteine amino acid residue following its 9^(th) amino acid residue atthe C-terminus. As set forth herein, references to amino acid positionscorrespond to the amino acid residues of Hep25. For example, referenceto amino acid position 9, corresponds to the 9^(th) amino acid residueof Hep25.

The peptides of the present invention bind ferroportin, preferably humanferroportin. Preferred peptides of the present invention specificallybind human ferroportin. As used herein, “specifically binds” refers to aspecific binding agent's preferential interaction with a given ligandover other agents in a sample. For example, a specific binding agentthat specifically binds a given ligand, binds the given ligand, undersuitable conditions, in an amount or a degree that is observable overthat of any nonspecific interaction with other components in the sample.Suitable conditions are those that allow interaction between a givenspecific binding agent and a given ligand. These conditions include pH,temperature, concentration, solvent, time of incubation, and the like,and may differ among given specific binding agent and ligand pairs, butmay be readily determined by those skilled in the art.

The peptides of the present invention that mimic the hepcidin activityof Hep25, the bioactive human 25-amino acid form, are herein referred toas “mini-hepcidins”. As used herein, a compound having “hepcidinactivity” means that the compound has the ability to lower plasma ironconcentrations in subjects (e.g. mice or humans), when administeredthereto (e.g. parenterally injected or orally administered), in adose-dependent and time-dependent manner. See e.g. as demonstrated inRivera et al. (2005), Blood 106:2196-9.

In some embodiments, the peptides of the present invention have in vitroactivity as assayed by the ability to cause the internalization anddegradation of ferroportin in a ferroportin-expressing cell line astaught in Nemeth et al. (2006) Blood 107:328-33. In vitro activity maybe measured by the dose-dependent loss of fluorescence of cellsengineered to display ferroportin fused to green fluorescent protein asin Nemeth et al. (2006) Blood 107:328-33. Aliquots of cells areincubated for 24 hours with graded concentrations of a referencepreparation of Hep25 or a mini-hepcidin. As provided herein, the EC₅₀values are provided as the concentration of a given compound (e.g.peptide) that elicits 50% of the maximal loss of fluorescence generatedby the reference Hep25 preparation. EC₅₀ of Hep25 preparations in thisassay range from 5 to 15 nM and preferred mini-hepcidins have EC₅₀values in in vitro activity assays of about 1,000 nM or less.

Other methods known in the art for calculating the hepcidin activity andin vitro activity of peptides according to the present invention may beused. For example, the in vitro activity of compounds may be measured bytheir ability to internalize cellular ferroportin, which is determinedby immunohistochemistry or flow cytometry using antibodies whichrecognizes extracellular epitopes of ferroportin. Alternatively, the invitro activity of compounds may be measured by their dose-dependentability to inhibit the efflux of iron from ferroportin-expressing cellsthat are preloaded with radioisotopes or stable isotopes of iron, as inNemeth et al. (2006) Blood 107:328-33.

Design of Mini-Hepcidins

Previous studies indicate that the N-terminal segment of Hep25 isimportant for its hepcidin activity and is likely to form the contactinterface with ferroportin. However, the importance of each N-terminalamino acid to hepcidin activity was unknown. Therefore, alanine-scanningmutagenesis was performed on residues 1-6 of Hep25 to determine thecontribution of each N-terminal amino acid to hepcidin activity. Asshown in FIG. 1, the T2A substitution did not substantially impacthepcidin activity. Phenylalanine substitutions (F4A or F9A) caused thelargest decrease, more than about 70%, in hepcidin activity. Theremaining alanine substitutions had detectable decreases in hepcidinactivity which were not as significant as the F4A or F9A substitutions.

To determine whether the highly conserved and apparently structurallyimportant F4 phenylalanine is important for hepcidin activity, the F4amino acid of Hep25 was systematically substituted with other aminoacids. As shown in FIG. 2A, making the side-chain more polar (F4Y) ledto substantial loss of hepcidin activity as did the substitution withD-phenylalanine (f) or charged amino acids (D, K and Y). However,hepcidin activity was maintained when the F4 residue was substitutedwith nonaromatic cyclohexylalanine, thereby indicating that a bulkyhydrophobic residue is sufficient for activity.

To determine whether the highly conserved and apparently structurallyimportant F9 phenylalanine is important for hepcidin activity, the F9amino acid of Hep25 was substituted with other amino acids. As shown inFIG. 2B, hepcidin activity not only decreased when F9 was substitutedwith alanine, but also when it was substituted with nonaromaticcyclohexylalanine, thereby indicating that an aromatic residue may beimportant for activity.

Mutational studies indicate that C326, the cysteine residue at position326 of human ferroportin, is the critical residue involved in bindinghepcidin. Thus, various N-terminal fragments of Hep25 containing athiol, i.e. Hep 4-7, Hep3-7, Hep3-8, Hep3-9, Hep1-7, Hep1-8, Hep1-9, andHep1-10 C7A, were chemically synthesized, refolded and their activitiesrelative to Hep25 were assayed using flow-cytometric quantitation of theferroportin-GFP degradation, iron efflux estimation based onmeasurements of cellular ferritin, and radioisotopic iron effluxstudies. The sequences and EC₅₀'s of these N-terminal fragments areshown in Table 1.

Remarkably and unexpectedly, as shown in FIG. 3, Hep1-9 and Hep1-10 C7Awere found to be quite active in the flow-cytometry assay offerroportin-GFP internalization. On a mass basis, Hep1-9 and Hep1-10 C7Awere only about 4-times less potent and on a molar basis, about 10-timesless potent than Hep25. Thus, Hep1-9 and Hep1-10 C7A were used as thebasis to construct other peptides having hepcidin activity.

To determine the importance of the cysteine thiol on the hepcidinactivity of Hep1-9, the C7 residue of Hep1-9 was substituted with aminoacids that have a similar shape but cannot form disulfide bonds to giveHep9-C7S (serine substitution) and Hep9C7-tBut (t-butyl-blockedcysteine) or with a cysteine modified by disulfide coupled tertiarybutyl, which can participate in disulfide exchange with HS-t-butyl asthe leaving group, to give Hep9C7-SStBut. As shown in FIG. 4, amino acidsubstitutions that ablated the potential for disulfide formation orexchange caused a complete loss of hepcidin activity, thereby indicatingthat disulfide formation is required for activity. Other C7 amino acidsubstitutions and their resulting hepcidin activities are shown in Table1.

Other peptides based on Hep1-9 and Hep1-10 C7A were constructed to bedisulfide cyclized, have unnatural amino acid substitutions, beretroinverted, have modified F4 and F9 residues, or have a positivecharge. The C-terminal amino acid was the amidated form. Themodifications and the resulting hepcidin activities are shown in Table1.

As shown in Table 1, with the exception of PR40 and PR41, mini-hepcidinswhich exhibit EC₅₀'s of about 1000 nM or less contain at least 6contiguous amino acid residues which correspond to residues 3-8 of Hep25(see Hep3-8). Thus, in some embodiments, preferred mini-hepcidins haveat least 6 contiguous amino acid residues that correspond to 6contiguous amino acid residues of Hep1-9, preferably residues 3-8. Theamino acid residues may be unnatural or uncommon amino acids, L- orD-amino acid residues, modified residues, or a combination thereof.

In some embodiments, the mini-hepcidins of the present invention have atleast one amino acid substitution, a modification, or an addition.Examples of amino acid substitutions include substituting an L-aminoacid residue for its corresponding D-amino acid residue, substituting aCys for homoCys, Pen, (D)Pen, Inp, or the like, substituting Phe forbhPhe, Dpa, bhDpa, Bip, 1Nal, and the like. The names and the structuresof the substituting residues are exemplified in Table 2. Other suitablesubstitutions are exemplified in Table 1. Examples of a modificationinclude modifying one or more amino acid residues such that the peptideforms a cyclic structure, retroinversion, and modifying a residue to becapable of forming a disulfide bond. Examples of an addition includeadding at least one amino acid residue or at least one compound toeither the N-terminus, the C-terminus, or both such as that exemplifiedin Table 1.

As shown in Table 1, a majority of the mini-hepcidins which exhibitEC₅₀'s of about 100 nM or less contain at least one Dpa or bhDpa aminoacid substitution. Thus, in some embodiments, the mini-hepcidins of thepresent invention have at least one Dpa or bhDpa amino acidsubstitution.

In view of the alanine substitution data of FIG. 1, in some embodiments,the mini-hepcidins of the present invention may have an Ala at aminoacid positions other than amino acid position 4 and 9 as long as thereis an available thiol for forming a disulfide bond at amino acidposition 7. See Hep9F4A and Hep9C-SStBut in Table 1.

In view of the position 4 amino acid substitution data of FIG. 2 andTable 1, the mini-hepcidins of the present invention may have an aminoacid substitution at position 4 which does not result in a substantialchange of its charge or polarity as compared to that of Hep25, Hep1-9 orHep1-10 C7A. Preferred amino acid substitutions at position 4 of Hep1-9or Hep1-10 C7A include Phe, D-Phe, bhPhe, Dpa, bhDpa, Bip, 1Nal, or thelike.

The original mini-hepcidins as referenced herein have the followingStructural Formula I

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10  I

wherein

-   -   A1 is Asp, Glu, pyroglutamate, Gln, Asn, or an unnatural amino        acid commonly used as a substitute thereof;    -   A2 is Thr, Ser, Val, Ala, or an unnatural amino acid commonly        used as a substitute thereof;    -   A3 is His, Asn, Arg, or an unnatural amino acid commonly used as        a substitute thereof;    -   A4 is Phe, Leu, Ile, Trp, Tyr, or an unnatural amino acid        commonly used as a substitute thereof which includes        cyclohexylalanine;    -   A5 is Pro, Ser, or an unnatural amino acid commonly used as a        substitute thereof;    -   A6 is Ile, Leu, Val, or an unnatural amino acid commonly used as        a substitute thereof;    -   A7 is Cys, Ser, Ala, or an unnatural amino acid commonly used as        a substitute thereof which includes S-tertiary butyl-cysteine;    -   A8 is Ile, Leu, Thr, Val, Arg, or an unnatural amino acid        commonly used as a substitute thereof;    -   A9 is Phe, Leu, Ile, Tyr, or an unnatural amino acid commonly        used as a substitute thereof which includes cyclohexylalanine;        and    -   A10 is Cys, Ser, Ala, or an unnatural amino acid commonly used        as a substitute thereof;    -   wherein the carboxy-terminal amino acid is in amide or        carboxy-form;    -   wherein a Cys or another sulfhydryl amino acid is present as one        of the amino acids in the sequence; and    -   wherein A1, A2, A3, A1 to A2, A1 to A3, A10, A9 to A10, A8 to        A10, or a combination thereof are optionally absent.

In some embodiments, A1 is Asp; A2 is Thr; A3 is His; A4 is Phe; A5 isPro; A6 is Ile; A7 is Ala; A8 is Ile; A9 is Phe; and A10 is Cys in amideform; wherein A1 or A1 to A2 are optionally absent.

In some embodiments, A1 is Asp, A2 is Thr, A3 is His, A4 is Phe, A5 isPro, A6 is Ile, A7 is Cys or an unnatural thiol amino acid, A8 is Ile,A9 is Phe in amide form, and A10 is absent.

In some embodiments, A1 and A2 are absent, A3 is His, A4 is Phe, A5 isPro, A6 is Ile, A7 is Cys or an unnatural thiol amino acid, A8 is Ile inamide form, and A9 and A10 are absent.

In some embodiments, A1 and A2 are absent, A3 is His, A4 is Phe, A5 isPro, A6 is Ile, A7 is Cys or an unnatural thiol amino acid in amideform, and A8 to A10 are absent.

In some embodiments, the unnatural amino acid of A1, A2, A3, A4, A5, A6,A7, A8, A9, A10, or a combination thereof is the corresponding D-aminoacid. For example, for A1, the unnatural amino acid may be D-Asp, D-Glu,D-Gln, D-Asn, or the like.

In some embodiments, the unnatural amino acid for:

-   -   A1 is D-Asp, D-Glu, D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida,        or N-MeAsp;    -   A2 is D-Thr, D-Ser, D-Val, Tle, Inp, Chg, bhThr, or N-MeThr;    -   A3 is D-His, D-Asn, D-Arg, Dpa, (D)Dpa, or 2-aminoindan;    -   A4 is D-Phe, D-Leu, D-Ile, D-Trp, Phg, bhPhe, Dpa, Bip, 1Nal,        bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine;    -   A5 is D-Pro, D-Ser, Oic, bhPro, trans-4-PhPro, cis-4-PhPro,        cis-5-PhPro, Idc;    -   A6 is D-Ile, D-Leu, Phg, Chg, Amc, bhIle, Ach, and N-MeIle;    -   A7 is D-Cys, D-Ser, D-Ala, Cys(S-tBut), homoCys, Pen, (D)Pen,        Dap(AcBr), and Inp;    -   A8 is D-Ile, D-Leu, D-Thr, D-Val, D-Arg, Chg, Dpa, bhIle, Ach,        or N-MeIle;    -   A9 is D-Phe, D-Leu, D-Ile, PheF5, N-MePhe, benzylamide, bhPhe,        Dpa, Bip, 1Nal, bhDpa, cyclohexylalanine; and    -   A10 is D-Cys, D-Ser, D-Ala.

In some embodiments, the amino acid substitution (and addition, ifindicated) for:

-   -   A1 is Ala, D-Ala, Cys, D-Cys, Phe, D-Phe, Asp or D-Asp linked to        Cys or D-Cys, Phe or D-Phe linked to a PEG molecule linked to        chenodeoxycholate, ursodeoxycholate, or palmitoyl, or Dpa or        (D)Dpa linked to palmitoyl;    -   A2 is Ala, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, or D-Gly;    -   A3 is Ala, D-Ala, Cys, D-Cys, Dpa, Asp or D-Asp linked to Dpa or        (D)Dpa;    -   A4 is Ala, D-Ala, Pro, or D-Pro;    -   A5 is Ala, D-Ala, Pro, D-Pro, Arg, D-Arg;    -   A6 is Ala, D-Ala, Phe, D-Phe, Arg, D-Arg, Cys, D-Cys;    -   A7 is His, or D-His;    -   A8 is Cys, or D-Cys; and    -   A9 is Phe or D-Phe linked to RA, Asp, D-Asp, Asp or D-Asp linked        to RB, bhPhe linked to RC, or cysteamide, wherein RA is        —CONH₂—CH₂—CH₂—S, -D-Pro linked to Pro-Lys or Pro-Arg, -bhPro        linked to Pro linked to Pro-Lys or Pro-Arg, -D-Pro linked to        bhPro-Lys or bhPro-Arg, wherein RB is        -PEG11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, -(PEG11)-(GPHyp)10, and        wherein RC is -D-Pro linked to Pro-Lys or Pro-Arg, -D-Pro linked        to bhPro-Lys or bhPro-Arg.

In some embodiments, the mini-hepcidin is a 10-mer sequence wherein A7is Ala and A10 is Cys.

In some embodiments, the mini-hepcidin forms a cyclic structure by adisulfide bond.

In some embodiments, the mini-hepcidin is a retroinverted peptide suchthat A1 is the C-terminus and A10 is the N-terminus and the amino acidresidues are D-amino acids. In some embodiments, the retroinvertedpeptide has at least one addition at the N-terminus, C-terminus, orboth. In some embodiments, the retroinverted peptide contains at leastone L-amino acid.

In some embodiments, the mini-hepcidin has an amino acid substitution atposition 4, position 9, or both. In some embodiments, the amino acidsubstituent is Phg, Phe, D-Phe, bhPhe, Dpa, Bip, 1Nal, Dpa, bhDpa, Amc,or cysteamide.

In some embodiments, the mini-hepcidin has an amino acid substitution atposition 7. In some embodiments, the amino acid substituent isCys(S-tBut), Ala, D-Ala, Ser, D-Ser, homoCys, Pen, (D)Pen, His, D-His,or Inp.

Examples of some original mini-hepcidins are provided in Table 1.

TABLE 1 EC₅₀ 1 2 3 4 5 6 7 8 9 10 (nM) Name Hep25      10 DTHFPICIFCCGCCHRSKCGMCCKT (SEQ ID NO: 1) Hep10wt D T H F P I C I F C (SEQ ID NO: 2)Length Hep4 (Hep4-7) - - - F P I C - - - >10,000 (SEQ ID NO: 3)Hep5 (Hep3-7) - - H F P I C - - - >10,000 (SEQ ID NO: 4)Hep6 (Hep3-8) - - H F P I C I - -    1000 (SEQ ID NO: 5)Hep7ΔDT (Hep3-9) - - H F P I C I F -     700 (SEQ ID NO: 6)Hep7 (Hep1-7) D T H F P I C - - - >10,000 (SEQ ID NO: 7) Hep8 (Hep1-8) DT H F P I C I - -    2000 (SEQ ID NO: 8) Hep9 (Hep1-9) D T H F P I C IF -      76 (SEQ ID NO: 9) Hep10 D T H F P I A I F C     100(Hep1-10 C7A) (SEQ ID NO: 10) Thiol Modified Hep9F4A D T H A P I C I F -  >3000 (SEQ ID NO: 11) Hep9C7-SStBut D T H A P I C-S-tBut I F -     700Hep9C7-tBut D T H A P I C-tBut I F - >10,000 Hep9-C7A D T H F P I A IF - >10,000 (SEQ ID NO: 12) Hep9-C7S D T H F P I S I F - >10,000(SEQ ID NO: 13) (D)C D T H F P I C I F -    1000 homoC D T H F P IhomoCys I F -     900 Pen D T H F P I Pen I F -     700 (D)Pen D T H F PI (D)Pen I F -    3000 Dap(AcBr) D T H F P I Dap(AcBr) I F -  >10000Disulfide Cyclized Cyc-1 C-D T H F P I C I F -     300 (SEQ ID NO: 14)Cyc-4 D T H F P I C I F-R1 -  >10000 Cyc-2 - C H F P I C I F -  >10000(SEQ ID NO: 15) Cyc-3 - - H F P I C I F-R1 -  >10000 Unnatural AA's PR10D Tle H Phg Oic Chg C Chg F -   >3000 PR11 D Tle H P Oic Chg C Chg F -  >3000 Retroinverted PR12 F I C I P F H T D -     900* riHep7ΔDT F I CI P F H - - -     150* Modified Retroinverted PR23 R2-F I C I P F H TD -     100 PR24 R3-F I C I P F H T D -    1000* PR25 F I C I P F H TD-R6 -     600 PR26 F I C I P F H T D-R6 - >10,000 PR27 R4-F I C I P F HT D -      20* PR28 R5-F I C I P F H T D -    3000 Modified F4 and F9F4bhPhe D T H bhPhe P I C I F -     700 F4Dpa D T H Dpa P I C I F -     30 F4Bip D T H Bip P I C I F -     150 F4 1Nal D T H 1Nal P I C IF -     110 F4bhDpa D T H bhDpa P I C I F -      80 F9bhPhe D T H F P IC I bhPhe -     150 F9Dpa D T H F P I C I Dpa -      70 F9Bip D T H F PI C I Bip -     150 F91Nal D T H F P I C I 1Nal -     200 F9bhDpa D T HF P I C I bhDpa -     100 PR39 D T H Dpa P I C I Dpa -      35 PR40 D -Dpa - P I C I F -      70 PR41 D - Dpa - P I C I Dpa -     300 PR43 D TH Dpa P R C R Dpa -     200 PR44 D T H Dpa Oic I C I F -      30 PR45 DT H Dpa Oic I C I Dpa -     150 PR46 D T H Dpa P C C C Dpa -      80Positive Charge PR13 D T H F P I C I F-R8 -     100 PR14 D T H F P I C IF-R9 -      90 PR15 D T H F P I C I F-R10 -     150 PR16 D T H F P I C IF-R11 -      50 PR17 D T H F P I C I F-R12 -     300 PR18 D T H F P I CI F-R13 -    1000 PR19 D T H F P I C I bhPhe-R8 -     700 PR20 D T H F PI C I bhPhe-R9 -     200 PR21 D T H F P I C I bhPhe-R12 -     500 PR22 DT H F P I C I bhPhe-R13 -     600 PR-1 C Inp (D)Dpa Amc R Amc Inp DpaCysteamide** -    1500 PR-2 C P (D)Dpa Amc R Amc Inp Dpa Cysteamide** -   2000 PR-3 C P (D)Dpa Amc R Amc Inp Dpa Cysteamide** -    1000 PR-4 CG (D)Dpa Amc R Amc Inp Dpa Cysteamide** -    2000 R1 = -CONH₂-CH₂-CH₂-SR2 = Chenodeoxycholate-(D)Asp-(PEG11)- R3 =Ursodeoxycholate-(D)Asp-(PEG11)- R4 = Palmitoyl-(PEG11)- R5 =(Palmitoyl)₂-Dap-PEG11-, wherein “Dap” = diaminopropionic acid R6 =-(PEG11)-GYIPEAPRDGQAYVRKDGEWVLLSTFL R7 = -(PEG11)-(GPHyp)10, “GPHyp” =Gly-Pro-hydroxyproline R8 = -PPK R9 = -PPR R10 = -bhProPK R11 = -bhProPRR12 = -PbhProK R13 = -PbhProR Underlined residues = D amino acids “-”indicates a covalent bond, e.g. point of attachment to the given peptideDouble underlined = residues connected by a disulfide link to form acyclized structure *active in vivo **oxidized The PEG compound may bePEG11, i.e. O-(2-aminoethyl)-O′(2-carboxyethyl)-undecaethyleneglycolPR12, riHep7ΔDT, PR23, PR24, PR25, PR26, PR27 and PR28 are retroinvertedmini-hepcidins and are shown, left to right, from their C-terminus totheir N-terminus.

TABLE 2 Uncommon or Unnatural Amino Acids Chg

L-α-cyclohexylglycine Tle

L-tert-leucine bhPhe

β-homophenylalanine Dpa

3,3-diphenyl-L-alanine bhPro

L-β-homoproline Phg

L-phenylglycine 1Nal

(1-naphthyl)-L-alanine bhDpa

(S)-3-Amino-4,4-diphenylbutanoic acid Bip

L-biphenylalanine Pen

L-Penicillamine (D)Pen

D-Penicillamine Cys(tBut)

S-t-butyl-L-cysteine Oic

octahydroindole-2-carboxylic acid Dap(AcBr)

N^(γ)-(bromoacetyl)-L-2,3-diaminopropionic acid homoCys

L-homocysteine Cys(S-tBut)

S-t-Butylthio-L-cysteine Amc

4-(aminomethyl)cyclohexane carboxylic acid Inp

isonipecotic acid bhAsp

Ida

N-MeAsp

N-MeThr

2-Aminoindane

PheF5

hPhe

Igl

trans-4-PhPro

cis-4-PhPro

cis-5-PhPro

Idc

bhIle

Ach

N-Melle

N-MePhe

Benzylamide

(D)Dpa

3,3-diphenyl-D-alanine Ahx

N-MeArg

2Nal

L-His(π-Me)

L-His(τ-Me)

Peptide Synthesis

Hep25 was synthesized at the UCLA Peptide Synthesis Core Facility usingsolid phase 9-fluorenylmethyloxycarbonyl (fmoc) chemistry. Specifically,the peptides were synthesized on an ABI 431A peptide synthesizer (PEBiosystems, Applied Biosystems, Foster City, Calif.) using fmoc aminoacids, Wang resin (AnaSpec, San Jose, Calif.), and double coupling forall residues. After cleavage, 30 mg crude peptides was reduced with1000-fold molar excess of dithiothreitol (DTT) in 0.5 M Tris buffer (pH8.2), 6 M guanidine hydrochloride, and 20 mM EDTA at 52° C. for 2 hours.Fresh DTT (500-molar excess) was added and incubated for an additionalhour at 52° C. The reduced peptides were purified on the 10-g C18SEP-PAK cartridges (Waters, Milford, Mass.) equilibrated in 0.1% TFA andeluted with 50% acetonitrile. The eluates were lyophilized andresuspended in 0.1% acetic acid. The reduced peptides were furtherpurified by reversed-phase high-performance liquid chromatography(RP-HPLC) on VYDAC C18 column (218TP510; Waters) equilibrated with 0.1%trifluoroacetic acid and eluted with an acetonitrile gradient. Theeluates were lyophilized, dissolved in 0.1% acetic acid, 20% DMSO, tothe approximate concentration of 0.1 mg/ml (pH 8), and air oxidized bystirring for 18 hours at room temperature. The refolded peptides werealso purified sequentially on the 10-g C18 SEP-PAK cartridge and on theRP-HPLC VYDAC C18 column using an acetonitrile gradient. The eluateswere lyophilized and resuspended in 0.016% HCl. The conformation ofrefolded synthetic hepcidin derivatives was verified by electrophoresisin 12.5% acid-urea polyacrylamide gel electrophoresis (PAGE), andpeptide masses were determined by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS;UCLA Mass Spectrometry Facility, Los Angeles, Calif.).

The other peptides set forth in Table 1 were synthesized by the solidphase method using either Symphony® automated peptide synthesizer(Protein Technologies Inc., Tucson, Ariz.) or CEM Liberty automaticmicrowave peptide synthesizer (CEM Corporation Inc., Matthews, N.C.),applying 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry (Fields & Noble(1990) Int J Pept Protein Res 35:161-214) and commercially availableamino acid derivatives and reagents (EMD Biosciences, San Diego, Calif.and Chem-Impex International, Inc., Wood Dale, Ill.). Peptides werecleaved from resin using modified reagent K (TFA 94% (v/v); phenol, 2%(w/v); water, 2% (v/v); TIS, 2% (v/v); 2 hours) and precipitated byaddition of ice-cold diethyl ether. Subsequently, peptides were purifiedby preparative reverse-phase high performance liquid chromatography(RP-HPLC) to >95% homogeneity and their purity evaluated bymatrix-assisted laser desorption ionization spectrometry (MALDI-MS, UCLAMass Spectrometry Facility, Los Angeles, Calif.) as well as analyticalRP-HPLC employing Varian ProStar 210 HPLC system equipped with ProStar325 Dual Wavelength UV-Vis detector with the wavelengths set at 220 nmand 280 nm (Varian Inc., Palo Alto, Calif.). Mobile phases consisted ofsolvent A, 0.1% TFA in water, and solvent B, 0.1% TFA in acetonitrile.Analyses of peptides were performed with a reversed-phase C18 column(Vydac 218TP54, 4.6×250 mm, Grace, Deerfield, Ill.) applying lineargradient of solvent B from 0 to 100% over 100 min (flow rate: 1 ml/min).

Other methods known in the art may be used to synthesize or obtain thepeptides according to the present invention. All peptides weresynthesized as carboxyamides (—CONH₂) which creates a charge-neutral endmore similar to a peptide bond than the negatively charged —COOH end.Nevertheless, peptides having the negatively charged —COOH end arecontemplated herein.

Activity Assays

Flow Cytometry.

The activity of peptides of the present invention was measured by flowcytometry as previously described. See Nemeth et al. (2006) Blood107:328-333, which is herein incorporated by reference. ECR293/Fpn-GFP,a cell line stably transfected with a ponasterone-inducible ferroportinconstruct tagged at the C-terminus with green fluorescent protein wasused. See Nemeth et al. (2004) Science 306:2090-2093, which is hereinincorporated by reference. Briefly, the cells were plated onpoly-D-lysine coated plates in the presence of 20 μM FAC, with orwithout 10 μM ponasterone. After 24 hours, ponasterone was washed off,and cells were treated with peptides for 24 hours. Cells were thentrypsinized and resuspended at 1×10⁶ cells/ml, and the intensity ofgreen fluorescence was analyzed by flow cytometry. Flow cytometry wasperformed on FAC SCAN (fluorescence activated cell scanner) AnalyticFlow Cytometer (Becton Dickinson, San Jose, Calif.) with CELLQUESTversion 3.3 software (Becton Dickinson). Cells not induced withponasterone to express Fpn-GFP were used to establish a gate to excludebackground fluorescence. Cells induced with ponasterone, but not treatedwith any peptides, were used as the positive control. Each peptide wastested over the range of concentrations (0, 0.01, 0.03, 0.1, 0.3, 1, 3and 10 μM). Each peptide treatment was repeated independently 3 to 6times. For each concentration of peptide, the results were expressed asa fraction of the maximal activity (F_(Hep25)) of Hep25 (in the doserange 0.01-10 μM), according to the formula1−((F_(x)−F_(Hep25))/(F_(untreated)−F_(Hep25))), where F was the mean ofthe gated green fluorescence and x was the peptide. The IEC₅₀concentrations are set forth in the Table 1.

Ferritin Assay.

Cells treated with peptides having hepcidin activity will retain ironand contain higher amounts of ferritin. Thus, following ferritin assaymay be used to identify mini-hepcidins according to the presentinvention. Briefly, HEK293-Fpn cells are incubated with 20 μM FAC withor without 10 μM ponasterone. After 24 hours, ponasterone is washed off,and hepcidin derivatives are added for 24 hours in the presence of 20 μMFAC. Cellular protein is extracted with 150 mM NaCl, 10 mM EDTA, 10 mMTris (pH 7.4), 1% Triton X-100, and a protease inhibitor cocktail(Sigma-Aldrich, St Louis, Mo.). Ferritin levels are determined by anenzyme-linked immunosorbent assay (ELISA) assay (Ramco Laboratories,Stafford, Tex., or Biotech Diagnostic, Laguna Niguel, Calif.) accordingto the manufacturer's instructions and are normalized for the totalprotein concentration in each sample, as determined by the bicinchoninicacid (BCA) assay (Pierce, Rockford, Ill.).

In Vivo Assays. Serum Iron Assay.

The decrease in serum iron after peptide administration is the principalmeasure of hepcidin activity. Thus, as provided herein, the hepcidinactivity of selected peptides of the present ^(i)nvention were assayedin vivo by measuring serum iron i^(n) test subjects. Briefly, C57/Bl6Jmice were maintained on NIH 31 rodent diet (333 parts per million (ppm)iron; Harlan Teklad, Indianapolis, Ind.). Two weeks before theexperiment, the mice were switched to a diet containing about 2-4 ppmiron ⁽Harlan Teklad, Indianapolis, Ind.) in order to suppress endogenoushepcidin. Peptide stocks were diluted to desired concentrations insterile phosphate buffered saline (PBS) or other diluents as describednext. Mice were subjected to the following treatments: (a) Injectedintraperitoneally either with 100 μl PBS (control) or with 50 μg peptidein 100 μl PBS; (b) Injected with 100 μl of peptide (or PBS) mixed with500 μg empty liposomes COATSOME EL series (NOF, Tokyo, Japan) (preparedas per manufacturer's recommendation); (c) Injected with 100 μl peptides(or PBS) solubilized with SL220 solubilization agent (NOF, Tokyo,Japan); (d) Gavaged with 250 μl of peptide (or PBS) in 1× solvent(Cremophor EL (Sigma)/ethanol/PBS; (12.5:12.5:75)). Mice were sacrificed4 hours later, blood was collected by cardiac puncture, and serum wasseparated using MICROTAINER tubes (Becton Dickinson, Franklin Lakes,N.J.). Serum iron was determined by using a colorimetric assay(Diagnostic Chemicals, Oxford, Conn.), which was modified for themicroplate format so that 10 μl serum was used per measurement. Theresults were expressed as the percentage of decrease in serum iron whencompared with the average value of serum iron levels in PBS-treatedmice.

As shown in FIG. 5, intraperitoneal (i.p.) administration of 50 μg PR12per mouse in PBS caused a significant decrease in serum iron after 4hours, when compared to i.p. administration of PBS. The serum irondecrease was similar to that caused by i.p. injection of 50 μg of Hep25.Injection (i.p.) of Hep9 did not result in a serum iron decrease. PR12is a retroinverted form of Hep9, and is resistant to proteolysis becauseof the retroinverted structure. The experiment indicates that increasedproteolytic resistance improves the activity of mini-hepcidins.

As shown in FIG. 6, i.p. administration of 200 nmoles of riHep7ΔDT inPBS resulted in serum iron concentrations significantly lower than thoseachieved after injection of PBS, and also lower than i.p. injection of20 nmoles of Hep25. Administration of 20 nmoles of riHep7ΔDT slightlybut not significantly reduced serum iron concentrations. The experimentindicates that after i.p. injection peptides as small as 7 amino acidsare able to display activity comparable to Hep25.

As shown in FIG. 7, i.p. administration of 20 nmoles PR27 in PBS causeda serum iron decrease comparable to that caused by i.p. administrationof 20 nmoles Hep25. This indicated that mini-hepcidin can achievesimilar potency to Hep25 in vivo. Higher concentration of PR27 (200nmoles) caused even greater decrease in serum iron concentrations.

As shown in FIG. 8, i.p. administration of 20 nmoles PR27 in liposomalsolution also caused a serum iron decrease similar to that caused byi.p. administration of 20 nmoles Hep25. Administration of liposomalsolution by itself did not affect serum iron levels. The liposomalsolution was prepared by mixing 100 μl of PBS with 500 μg emptyliposomes COATSOME EL series (NOF, Tokyo, Japan) (prepared as permanufacturer's recommendation). Mini-hepcidin PR28 dissolved inliposomal solution, however, showed lesser ability to decrease serumiron than PR27. The experiment indicates that suspension of peptides inliposomes does not affect their activity. Thus, liposomes may be usefulfor oral administration of peptides according to the present invention.

As shown in FIG. 9, oral administration of PR27 200 nmoles by gavage ina cremophore EL solution caused a decrease in serum iron in mice ascompared to oral administration of PBS in the same formulation.Cremophor EL increases solubility of chemicals, and is frequently usedexcipient or additive in drugs. Cremophor EL solution was prepared bymixing Cremophor EL (Sigma), ethanol and PBS in a ratio 12.5:12.5:75.250 μl of the solution was administered by gavage to mice.

Thus, the present invention may be used to decrease serum iron insubjects. A preferred mini-hepcidin according to the present inventionis a retroinverted peptide which comprises a PEG molecule, such asPEG11, linked to its N-terminal amino acid. In some embodiments, the PEGmolecule is linked to palmitoyl group or diaminopropionic acid linked toone or more palmitoyl groups.

In addition to assaying the effect on serum iron content, other in vivoassays known in the art may be conducted to identify mini-hepcidinsaccording to the present invention and/or determine the therapeuticallyeffective amount of a given peptide or mini-hepcidin according to thepresent invention. Examples of such assays include the following:

Tissue Iron Assay.

In addition to or instead of the serum iron assay above, tissue irondistribution can be determined by enhanced Perl's stain of liver andspleen sections obtained from the treated mice. Briefly, the tissuesections are fixed in 4% paraformaldehyde/PBS, incubated in Perl'ssolution (1:1, 2% HCl and 2% potassium ferrocyanide) anddiaminobenzidine in 0.015% hydrogen peroxide. Tissue non-heme iron maybe quantitated using the micromethod of Rebouche et al. (2004) J BiochemBiophys Methods. 58(3):239-51; Pak et al. (2006) Blood 108(12):3730-5.100 mg pieces of liver and spleen are homogenized and acid is added torelease non-heme bound iron which is detected by colorimetric reactionusing ferrozine and compared to controls. Treatment with mini-hepcidinswould be expected to cause redistribution of iron from other tissues tothe spleen. Over weeks to months, the administration of mini-hepcidinswould be expected to decrease tissue iron content in all tissues becauseof diminished dietary iron absorption.

Hematology Assays.

Hematology assays may be used to identify mini-hepcidins according tothe present invention and/or determine the therapeutically effectiveamount of a given peptide or mini-hepcidin according to the presentinvention. Briefly, blood from treated subjects is collected intoheparin-containing tubes. Hemoglobin, RBC, MCV, EPO, white cellparameters, reticulocyte counts, and reticulocyte Hgb content aredetermined using methods known in the art and compared to controls.Treatment with mini-hepcidins would be expected to cause a decrease inMCV and diminish the Hgb content of reticulocytes. Administration ofmini-hepcidins in excessive amounts would be expected to decrease Hgb.

Iron Export Assays.

Iron (⁵⁵Fe) export assays known in the art using primary hepatocytes andmacrophages may be used to identify mini-hepcidins according to thepresent invention and/or determine the therapeutically effective amountof a given peptide or mini-hepcidin according to the present invention.Peptides having hepcidin activity will diminish or decrease the releaseof ⁵⁵Fe from cells. Briefly, cells are incubated with ⁵⁵Fe-NTA or⁵⁵Fe-Tf for 36 hours. After washing off unincorporated ⁵⁵Fe, cells aretreated with a given peptide or a control. In case of ferroportinmutants, the transfection is performed prior to addition of ⁵⁵Fe andexpression allowed to proceed during the 36 hour iron-loading period.Aliquots of the media are collected after 1, 4, 8, 24, 36, 48 and 72hours and radioactivity is determined by a scintillation counter.Cell-associated radioactivity can be measured by centrifuging cellsthrough silicone oil to lower the non-specific binding of radiolabelediron to cells using methods known in the art.

To determine whether a given peptide modifies the internalization anddegradation of endogenous ferroportin, the protein levels and cellulardistribution of ferroportin in hepatocytes and macrophages treated withthe peptide may be assayed using Western blotting, immunohistochemistryand ferroportin antibodies known in the art.

Modified Mini-Hepcidins

Additional mini-hepcidins according to the present invention are shownin Table 3 and Table 4 as follows:

TABLE 3 Active @ 20 1 2 3 4 5 6 7 8 9 10 EC₅₀ (nM) nmoles/mouse Name D TH F P I C I F C in vitro IP PR42′ D T H Dpa P R C R Dpa 30 PR47 D T HDpa P I C I F-R4 50 N PR48 D T H Dpa P I C I Dpa-R4 50 N PR49 H Dpa P IC I F-R4 <10 N PR50 H Dpa P I C I Dpa-R4 <10 N PR51 D T H Dpa P V C VF-R4 100 PR52 D T H Dpa P L C L F-R4 <10 PR53 N-MeAsp T H Dpa P I C IbhPhe-R14 10 PR54 N-MeAsp T H Dpa bhPro I C I bhPhe-R14 10 PR55 N-MeAspT H Dpa P Ach C Ach F-R14 10 PR56 N-MeAsp T H Dpa Oic R C R bhPhe-R14 15PR57 N-MeAsp T H Dpa bhPro R C R bhPhe-R14 2 Y PR58 Ida T H Dpa P I C IbhPhe-R14 1 PR59 Ida T H Dpa bhPro I C I bhPhe-R14 2 N PR60 Ida T H DpaP Ach C Ach F-R14 3 Y* PR61 Ida T H Dpa bhPro R C R bhPhe-R14 10-100 Y(also by SQ) R4 = Palmitoyl-(PEG11)-, PEG11 =O-(2-aminoethyl)-O′-(2-carboxyethyl)-undecaethyleneglycol R14=Palmitoyl-PEG-miniPEG3-, and “miniPEG3” =11-amino-3,6,9-trioxaundecanoic acid Underlined residues = D amino acids“—” indicates a covalent bond, e.g. point of attachment to the givenpeptide IP = intraperitoneal administration, SQ = subcutaneousadministration *= Exhibits detectable and reproducible activity at about50% lower than the most active compounds in Table 3 and 4. As usedherein, “Active” means that at 4 hours after injection serum iron inpeptide-injected mice decreased statistically significantly (p < 0.05)compared to solvent-injected mice. “Activity” refers to the % decreaseof serum iron 4 hours after injection compared to solvent-treated mice.In some embodiments, PEG11 can be substituted with miniPEG3 and miniPEG3can be substituted with PEG11.

TABLE 4 Active @ 20 1 2 3 4 5 6 7 8 9 10 nmoles/mouse Name D T H F P I CI F C IP SQ PR62 Ida T H Dpa bhPro R C R bhPhe-R14 N PR63 Ida T H DpabhPro N-MeArg C N-MeArg bhPhe-R14 Y PR64 Ida T H Dpa bhPro bhArg C bhArgbhPhe-R14 N PR65 Ida T H Dpa bhPro R C R bhPhe-R15 Y Y PR66 Ida T H DpabhPro R C R bhPhe Y N PR67 Ida T H Dpa bhPro R Cys(S-S- R bhPhe Y Y Pal)PR68 Ida T H Dpa bhPro R Cys(S-S- R bhPhe Y cysteamine- Pal) PR69 Ida TH Dpa bhPro R Cys(S-S- R bhPhe Y* Cys-NHPal) PR70 Ida T H Dpa bhPro RCys(S-S- R bhPhe-R14 Y* Cys) PR71 Ida(NHPal) T H Dpa bhPro R C R bhPheY* N PR72 Ida T H Dpa bhPro R C R bhPhe Ida(NHPal) Y N PR73 Ida T H DpabhPro R C R bhPhe Ahx- Y Ida(NHPal) PR74 Ida T H Dpa bhPro R C R bhPheAhx- Y Ida(NBzI2) PR75 Ida T H Dpa bhPro R C R bhPhe-R16 N PR76 D T H FP R Cys(S-S- R W-R17 N tBut) PR77 D T H F P R Cys(S-S- R W-R18 N tBut)PR78 D T H F P R Cys(S-S- R W-R19 N tBut) PR79 D T H F P R Cys(S-S- RW-R20 N tBut) PR82 Ida T H Dpa bhPro R C R W Ahx- Y Ida(NHPal) R4 =Palmitoyl-(PEG11)-, wherein PEG11 =O-(2-aminoethyl)-O′-(2-carboxyethyl)-undecaethyleneglycol R14 =Palmitoyl-PEG-miniPEG3-, and “miniPEG3” =11-amino-3,6,9-trioxaundecanoic acid R15 = Palmitoyl-PEG- R16 =S-(Palmityl)thioglycolic-PEG- R17 = Butanoyl-PEG11- R18 =Octanoyl-PEG11- R19 = Palmitoyl-PEG11- R20 = Tetracosanoyl-PEG11-Ahx-Ida(NHPal) = Aminohexanoic acid spacer between peptide residue 9 andIda residue; Palmitylamine amide on Ida side chain Ida(NHPal) =Palmitylamine amide on Ida side chain Ida(NBzI2) = N,N′-Dibenzylamineamide on Ida side chain Cys(S-S-Pal) = Palmitoyl attached to Cys7 via adisufide bond Cys(S-S-cysteamine-Pal) = Palmitoyl attached to Cys7 viaSS-Cysteamine Cys(S-S-Cys-NHPal) = Palmitylamine attached to Cys7 viaanother Cys Cys(S-S-Cys) = Cys attached to Cys7 via disulfide bondUnderlined residues = D amino acids “—” indicates a covalent bond, e.g.point of attachment to the given peptide *= detectable and reproducibleactivity but at least 50% lower than other active compounds in Table 4.As used herein, “Active” means that the injection of the compoundresulted in statistically significant (p < 0.05) lowering of serum ironcompared to solvent injection when measured 4 hours afteradministration. “Activity” refers to the % decrease of serum iron 4hours after injection compared to solvent-treated mice. IP =intraperitoneal administration, SQ = subcutaneous administration In someembodiments, PEG11 can be substituted with miniPEG3. In someembodiments, miniPEG3 can be substituted with PEG11. In someembodiments, PEG can be substituted with PEG11, but not miniPEG3.

In Tables 3 and 4, PR47, PR48, PR49, PR50, PR51, PR52, PR76, PR77, PR78,and PR79 are retroinverted mini-hepcidins and are shown, left to right,from their C-terminus to their N-terminus in order to exemplify thealignment between their amino acid residues and that of residues 1-10 ofHep25. Thus, the conventional recitation of these retroinvertedmini-hepcidins from their N-terminus to their C-terminus are as follows(D amino acids are underlined):

PR47: R4-F-I-C-I-P-Dpa-H-T-D

PR48: R4-Dpa-I-C-I-P-Dpa-H-T-D

PR49: R4-F-I-C-I-P-Dpa-H

PR50: R4-Dpa-I-C-I-P-Dpa-H

PR51: R4-F-V-C-V-P-Dpa-H-T-D

PR52: R4-F-L-C-L-P-Dpa-H-T-D

PR76: R17-W-R-Cys(S-S-tBut)-R-P-F-H-T-D

PR77: R18-W-R-Cys(S-S-tBut)-R-P-F-H-T-D

PR78: R19-W-R-Cys(S-S-tBut)-R-P-F-H-T-D

PR79: R20-W-R-Cys(S-S-tBut)-R-P-F-H-T-D

As shown in Table 4, the route of administration may play a role in theactivity of the given mini-hepcidin (compare, for example, PR65 andPR66). Thus, the indication of no activity of some of the mini-hepcidinsin Tables 3 and 4 should not be interpreted as indicating that the givenmini-hepcidin lacks any activity at any route of administration and/ordosage. In fact, as shown in Table 3, quite a few of such mini-hepcidinsexhibit significant in vitro activity at considerably lower dosages asthe original mini-hepcidins.

These additional mini-hepcidins are modifications of the mini-hepcidinsas set forth in PCT/US2009/066711 (hereinafter referred to as “originalmini-hepcidins” and having the Structural Formula I). As used herein,mini-hepcidins which are modifications of the original mini-hepcidinsare referred to herein as “modified mini-hepcidins”. As used herein,“mini-hepcidins” refers to both original mini-hepcidins and modifiedmini-hepcidins. Modified mini-hepcidins according to the presentinvention have the following Structural Formula IA or IB:

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10  IA

A10-A9-A8-A7-A6-A5-A4-A3-A2-A1  IB

-   A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D-pyroglutamate, Gln,    D-Gln, Asn, D-Asn, or an unnatural amino acid commonly used as a    substitute thereof such as bhAsp, Ida, Ida(NHPal), and N-MeAsp,    preferably Ida and N-MeAsp;-   A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile, Ala, D-Ala or    an unnatural amino acid commonly used as a substitute thereof such    as Tle, Inp, Chg, bhThr, and N-MeThr;-   A3 is His, D-His, Asn, D-Asn, Arg, D-Arg, or an unnatural amino acid    commonly used as a substitute thereof such as L-His(π-Me),    D-His(π-Me), L-His(τ-Me), or D-His(τ-Me);-   A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr, D-Tyr, or    an unnatural amino acid commonly used as a substitute thereof such    as Phg, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, Amc, PheF5, hPhe, Igl,    or cyclohexylalanine, preferably Dpa;-   A5 is Pro, D-Pro, Ser, D-Ser, or an unnatural amino acid commonly    used as a substitute thereof such as Oic, bhPro, trans-4-PhPro,    cis-4-PhPro, cis-5-PhPro, and Idc, preferably bhPro;-   A6 is Arg, D-Arg, Ile, D-Ile, Leu, D-Leu, Thr, D-Thr, Lys, D-Lys,    Val, D-Val, or an unnatural amino acid commonly used as a substitute    thereof such as D-Nω,ω-dimethyl-arginine, L-Nω,ω-dimethyl-arginine,    D-homoarginine, L-homoarginine, D-norarginine, L-norarginine,    citrulline, a modified Arg wherein the guanidinium group is modified    or substituted, Norleucine, norvaline, bhIle, Ach, N-MeArg, and    N-MeIle, preferably Arg;-   A7 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, or an unnatural amino acid    commonly used as a substitute thereof such as Cys(S-tBut), homoCys,    Pen, (D)Pen, preferably S-tertiary butyl-cysteine, Cys(S-S-Pal),    Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), and Cys(S-S-Cys) or any    amino acid derivative having an exchangeable cysteine;-   A8 is Arg, D-Arg, Ile, D-Ile, Leu, D-Leu, Thr, D-Thr, Lys, D-Lys,    Val, D-Val, or an unnatural amino acid commonly used as a substitute    thereof such as D-Nω,ω-dimethyl-arginine, L-Nω,ω-dimethyl-arginine,    D-homoarginine, L-homoarginine, D-norarginine, L-norarginine,    citrulline, a modified Arg wherein the guanidinium group is modified    or substituted, Norleucine, norvaline, bhIle, Ach, N-MeArg, and    N-MeIle, preferably Arg;-   A9 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Tyr, D-Tyr, Trp, D-Trp,    Phe-R^(a), D-Phe-R^(a), Dpa-R^(a), D-Dpa-R^(a), Trp-R^(a),    bhPhe-R^(a), or an unnatural amino acid commonly used as a    substitute thereof such as PheF5, N-MePhe, benzylamide,    2-aminoindane, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, and    cyclohexylalanine, which may or may not have R^(a) linked thereto,    preferably bhPhe and bhPhe-R^(a), wherein R^(a) is palmitoyl-PEG-,    wherein PEG is PEG11 or miniPEG3, palmitoyl-PEG-PEG, wherein PEG is    PEG11 or miniPEG3, butanoyl (C4)-PEG11-, octanoyl (C8,    Caprylic)-PEG11-, palmitoyl (C16)-PEG11-, or tetracosanoyl (C24,    Lignoceric)-PEG11-; and-   A10 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, or an unnatural amino    acid such as Ida, Ida(NHPal)Ahx, and Ida(NBzl2)Ahx;    wherein the carboxy-terminal amino acid is in amide or carboxy-form;    wherein at least one sulfhydryl amino acid is present as one of the    amino acids in the sequence (and preferably, the sulfydryl group is    capable of exchange); and    wherein A1, A1 to A2, A10, or a combination thereof are optionally    absent, with the proviso that the peptide is not one of the peptides    as set forth in Table 1. In some embodiments, the modified    mini-hepcidin forms a cyclic structure by a disulfide bond. In some    embodiments, the N-terminal amino acid is a free amine. In some    embodiments, the N-terminal amino acid is a blocked amine such as    with an acetyl group. In some embodiments, A6 and/or A8 is a lysine    derivative such as N-ε-Dinitrophenyl-lysine, N-ε-Methyl-lysine,    N,N-ε-Dimethyl-lysine, and N,N,N-ε-Trimethyl-lysine.

Five of the modified mini-hepcidins in Table 4 contain tryptophan inorder to facilitate measurements of their concentration. Thus, in someembodiments, the modified mini-hepcidins of the present inventioncontain tryptophan. In some embodiments, the tryptophan residue(s) isdeleted or substituted with another amino acid. Other modifiedmini-hepcidins were made by modifying original mini-hepcidins bysubstituting the two isoleucines flanking the cysteine with arginines orarginine derivatives. Unexpectedly, it was found that substituting theseisoleucines with arginines resulted in mini-hepcidins with increasedactivity. It is believed that the unexpectedly superior activity is theresult of the presence of arginine at the 6th amino acid position and/orthe 8th amino acid position. Thus, in some embodiments, the amino acidresidue at position A6 and/or A8 of structural formula I is arginine.Additional modifications of such mini-hepcidins also resulted inunexpectedly higher activities. The modified mini-hepcidins according tothe present invention unexpectedly exhibit superior activity as comparedto the modified mini-hepcidins exemplified in U.S. Ser. No. 13/131,792.In addition, it was found that many of the modified mini-hepcidinsretain their activity when subcutaneously administered.

In some embodiments the N-terminal amino acid has a free amine. In someembodiment the N-terminal amine is blocked with a group that removes itscharge, preferably an acetyl or formal group. In some embodiments theN-terminal amine is modified to be conjugated with an acyl chain withpreferred embodiments with a fatty acid such as caprylic, capric,lauric, myristic, palmitic, or stearic such that an acyl chain is on theN-terminus. The acyl chain also may be attached via a linker commonlyknown in the art, e.g. a polyethylene glycol linker, preferably PEG3.

In some embodiments, the side-chain of amino acid A1 can be modified asindicated for the N-terminal amine. For example, the free carbonyl groupon the amino acid A1 can be modified, e.g. blocked by an acyl group suchas palmitoyl (see PR71).

In some embodiment, A4 is a bulky hydrophobic amino acid such as Phe,Tyr, Trp, Leu, or Ile or any unnatural amino acid commonly used as asubstitute thereof that contains 4 or more carbons in its side-chain,preferably a cyclic structure such as Phg, Bip, 1Nal, 2Nal, Amc, PheF5,Igl (L-2-indanylglycine) or Cha (L-cyclohexylalanine), preferably Dpa.In some embodiments, A4 contains the betahomo form of the above bulkyhydrophobic amino acids, e.g. bhPhe, or bhDpa. Other modifications tothe side-chain include aromatic substituents such as those disclosed inWang et al. (2002) Tetrahedron 58:3101-3110 and Wang et al. (2002)Tetrahedron 58:7365-7374. In some embodiments, the A4 residue is aD-amino acid.

In some embodiments, the amino acid at position A9 is a bulkyhydrophobic amino acid such as Phe, Tyr, Trp, Leu, or Ile or anyunnatural amino acid commonly used as a substitute thereof that contains4 or more carbons in its side-chain, preferably a cyclic structure suchas Phg, Dpa, Bip, 1Nal, 2Nal, Amc, PheF5, Igl or Cha, such as a cyclicor aromatic group containing 1 or more rings or aromatic substituents.In some embodiments, the A9 residue is Dpa or Trp.

In some embodiments, the mini-hepcidins of the present invention aremodified or formulated in order to maintain and/or increase its in vivobioavailability. For example, in some embodiments, the peptide chain isconjugated with an acyl chain. In some embodiments, the acyl chain maybe conjugated to the N-terminal or C-terminal amino acid or a Cysteineresidue. In some embodiments, the acyl chain is conjugated to the A7residue. The acyl chain may include a fatty acid such as caprylic,capric, lauric, myristic, palmitic, or stearic. The acyl chain also mayalso contain a spacer such as a polyethylene glycol spacer. In someembodiments, the spacer is a polyethylene glycol spacer (1-11 PEG),preferably PEG3 or PEG11. In some embodiments, the spacer comprises ofan amino acid where the number of carbons between the amino group andthe carboxylic acid group is separated by about 2-8 carbons, such as6-aminohexanoic acid (see, for example, PR73). Other suitable spacersinclude a hydrophobic structure that has a ring and/or aromaticcharacter (see, for example, PR74).

The modified mini-hepcidins were demonstrated in a mouse hemochromatosismodel that daily administration of the modified mini-hepcidins, e.g.PR65, prevented iron overload. Therefore, the modified mini-hepcidinsaccording to the present invention, alone or in combination with one ormore original mini-hepcidins, may be administered to subjects in orderto treat, e.g. inhibit and/or reduce, iron overload in subjects, such ashumans. Therefore, modified and original mini-hepcidins according to thepresent invention may be used in medicaments and treatments in order totreat iron overload disorders, e.g. beta-thalassemia and hereditaryhemochromatosis, by inhibiting and/or reducing iron overload insubjects. In some embodiments, at least one modified mini-hepcidinand/or at least one original mini-hepcidin is administered to subjectsbefore, during, after, or a combination thereof, symptoms of ironoverload are observed and/or being diagnosed as having an iron overloaddisorder.

Thus, in some embodiments, one or more modified mini-hepcidins, alone orin combination with one or more original mini-hepcidins, are provided inthe form of a composition which comprises a carrier suitable for itsintended purpose. The compositions may also include one or moreadditional ingredients suitable for its intended purpose. For example,for assays, the compositions may comprise liposomes, niclosamide, SL220solubilization agent (NOF, Japan), cremophor EL (Sigma), ethanol, andDMSO. For treatment of an iron overload disease, the compositions maycomprise different absorption enhancers and protease inhibitors, solidmicroparticles or nanoparticles for peptide encapsulation (such aschitosan and hydrogels), macromolecular conjugation, lipidization andother chemical modification.

The present invention also provides kits comprising one or more modifiedmini-hepcidins, alone or in combination with one or more originalmini-hepcidins, and/or compositions of the present invention packagedtogether with reagents, devices, instructional material, or acombination thereof. For example, the kits may include reagents used forconducting assays, drugs and compositions for diagnosing, treating, ormonitoring disorders of iron metabolism, devices for obtaining samplesto be assayed, devices for mixing reagents and conducting assays, andthe like.

As the peptides of the present invention exhibit hepcidin activity, i.e.act as agonists of ferroportin degradation, one or more modifiedmini-hepcidins, alone or in combination with one or more originalmini-hepcidins, may be used to treat iron overload diseases. Forexample, one or more modified mini-hepcidins, alone or in combinationwith one or more original mini-hepcidins, may be administered to asubject to ameliorate the symptoms and/or pathology associated with ironoverload in iron-loading anemias (especially β-thalassemias) wherephlebotomy is contraindicated and iron chelators are the mainstay oftreatment but are often poorly tolerated. One or more modifiedmini-hepcidins, alone or in combination with one or more originalmini-hepcidins, may be used to treat hereditary hemochromatosis,especially in subjects who do not tolerate maintenance phlebotomy. Oneor more modified mini-hepcidins, alone or in combination with one ormore original mini-hepcidins, may be used to treat acute iron toxicity.In some embodiments, treatment with one or more modified mini-hepcidins,alone or in combination with one or more original mini-hepcidins, may becombined with phlebotomy or chelation.

Thus, one or more modified mini-hepcidins, alone or in combination withone or more original mini-hepcidins may be administered to a subject,preferably a mammal such as a human. In some embodiments, theadministration to the subject is before, during, and/or after thesubject exhibits an increase in iron levels and/or abnormally highlevels of iron. In some embodiments, the subject to be treated is onewho is at risk of having high levels of iron and/or has a geneticpredisposition to having an iron overload disease. In some embodiments,the peptides are administered in a form of a pharmaceutical composition.In some embodiments, the peptides are administered in a therapeuticallyeffective amount. As used herein, a “therapeutically effective amount”is an amount which ameliorates the symptoms and/or pathology of a givendisease of iron metabolism as compared to a control such as a placebo.

A therapeutically effective amount may be readily determined by standardmethods known in the art. The dosages to be administered can bedetermined by one of ordinary skill in the art depending on the clinicalseverity of the disease, the age and weight of the subject, or theexposure of the subject to iron. Preferred effective amounts ofmini-hepcidins range from about 0.01 to about 10 mg/kg body weight,about 0.01 to about 3 mg/kg body weight, about 0.01 to about 2 mg/kg,about 0.01 to about 1 mg/kg, or about 0.01 to about 0.5 mg/kg bodyweight for parenteral formulations. Effective amounts for oraladministration may be up to about 10-fold higher. Moreover, treatment ofa subject with a peptide or composition of the present invention caninclude a single treatment or, preferably, can include a series oftreatments. It will be appreciated that the actual dosages will varyaccording to the particular peptide or composition, the particularformulation, the mode of administration, and the particular site, host,and disease being treated. It will also be appreciated that theeffective dosage used for treatment may increase or decrease over thecourse of a particular treatment. Optimal dosages for a given set ofconditions may be ascertained by those skilled in the art usingconventional dosage-determination tests in view of the experimental datafor a given peptide or composition. Changes in dosage may result andbecome apparent by standard diagnostic assays known in the art. In someconditions chronic administration may be required.

The pharmaceutical compositions of the invention may be prepared in aunit-dosage form appropriate for the desired mode of administration. Thecompositions of the present invention may be administered for therapy byany suitable route including oral, rectal, nasal, topical (includingbuccal and sublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous and intradermal). A variety of administrationroutes can be used in accordance with the present invention, includingoral, topical, transdermal, nasal, pulmonary, transpercutaneous (whereinthe skin has been broken either by mechanical or energy means), rectal,buccal, vaginal, via an implanted reservoir, or parenteral. Parenteralincludes subcutaneous, intravenous, intramuscular, intraperitoneal,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques, as well as injectable materials (including polymers) forlocalized therapy. In some embodiments, the route of administration issubcutaneous. In some embodiments, the composition is in a sealedsterile glass vial. In some embodiments, the composition contains apreservative. Pharmaceutical compositions may be formulated as bulkpowder, tablets, liquids, gels, lyophilized, and the like, and may befurther processed for administration. See e.g. REMINGTON: THE SCIENCEAND PRACTICE OF PHARMACY. 20^(th) ed. (2000) Lippincott Williams &Wilkins. Baltimore, Md., which is herein incorporated by reference.

It will be appreciated that the preferred route will vary with thecondition and age of the recipient, the nature of the condition to betreated, and the chosen peptide and composition.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of at least one peptide as disclosedherein, and a pharmaceutically acceptable carrier or diluent, which maybe inert. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,bulking agent, coatings, antibacterial and antifungal agents,preservatives, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration and known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.

Supplementary compounds can also be incorporated into the compositions.Supplementary compounds include niclosamide, liposomes, SL220solubilization agent (NOF, Japan), Cremophor EL (Sigma), ethanol, andDMSO.

Toxicity and therapeutic efficacy of the peptides and compositions ofthe present invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Peptides whichexhibit large therapeutic indices are preferred. While peptides thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such peptides to the site of affectedtissue in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofpeptides of the present invention lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anypeptide used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography or bymass spectroscopy.

Modified Mini-Hepcidin, PR65

Peptide Synthesis.

Modified mini-hepcidins according to the present invention weresynthesized using standard solid-phase fmoc chemistry and was purifiedby reverse-phase HPLC. For PR65, from A1 to A9 of Structural Formula IA(i.e. from the N to C terminus) the primary sequence was all L-aminoacids as follows: iminodiacetic acid, threonine, histidine,diphenylalanine, beta-homo proline, arginine, cysteine, arginine andbeta-homo phenylalanine. The C-terminal carboxyamide was derivatizedwith a polyethylene glycol (PEG) linker and palmitic acid groups (FIG.10A). Human hepcidin was purchased from Peptides International(Louisville, Ky.).

Animal Studies.

All studies were approved by the UCLA Office of Animal ResearchOversight. Six weeks old male wild-type C57BL/6 mice were used tocompare the activities of native hepcidin and PR65, and to test theeffect of PR65 after intraperitoneal versus subcutaneous route ofadministration. Hepcidin and PR65 were administered in 100 μl of SL220,a PEG-phospholipid based solubilizer (NOF Corporation, Japan) (Preza GC, et al. (2011) J Clin. Invest. 121(12):4880-4888) and iron parameterswere measured after 4 hours. This solvent does not significantly changeserum iron concentrations in mice (<5 μM change, data not shown).

The therapeutic effects of PR65 was studied in hepcidin-1 knockout mice(Hamp1^(−/−)) (Lesbordes-Brion J C, et al. (2006) Blood108(4):1402-1405) and backcrossed onto the C57BL/6 background (N4, 99%gene marker identity) using marker-assisted accelerated backcrossing(Charles River Laboratories, Wilmington, Mass.). PR65 was administeredsubcutaneously in 100 μl of SL220 solubilizer. Short-term studies werecarried out for up to 48 hours to establish the effectiveness of asingle injection. Long-term studies (“prevention” and “treatment”) werecarried out for 2 weeks using daily injections and iron andhematological parameters measured 24 hours after the last injection.

To test the ability of PR65 to prevent, inhibit, or reduce iron loading(“prevention” studies), male Hamp1^(−/−) mice were iron-depleted byplacing them on a low-iron diet (4 ppm iron) for 2 months starting atthe age of 5-6 weeks. The regimen was developed to match the hepaticiron content of wild-type C57B6 mice, about 2-3 μmoles/g wet liver(Ramos E, et al. (2011) Hepatology 53(4):1333-1341). A group of mice wasanalyzed immediately after iron depletion (baseline group), and theremaining animals were switched to an iron-loading diet (standard chow,about 300 ppm Fe) and received daily subcutaneous injection of solventor PR65 (20, 50 or 100 nmoles) for 2 weeks. All mouse diets wereobtained from Harlan-Teklad (Madison, Wis.).

To test the effect of PR65 on iron-loaded Hamp1^(−/−) mice (“treatment”studies), male mice were kept on the standard diet for their entirelifespan. Beginning at 12-14 weeks of age, 50 nmoles of PR65 or solventwas injected daily by the subcutaneous route for 2 weeks.

Measurement of Iron and Hematological Parameters.

Serum iron and non-heme iron concentrations were determined aspreviously described (Ramos E, et al. (2011) Hepatology53(4):1333-1341), using acid treatment followed by a colorimetric assayfor iron quantitation (Genzyme, Cambridge, Mass.). Deparaffinizedsections were stained with the Perls Prussian blue stain for non-hemeiron, enhanced with the SG peroxidase substrate kit (Vector Labs,Burlingame, Calif.) and counterstained with nuclear fast red. Completeblood counts were obtained with a HemaVet blood analyzer (DrewScientific, Oxford, Conn.).

Statistical Analysis.

The statistical significance of differences between group means wasevaluated using Student T-test and the Sigmaplot 11.0 package (SystatSoftware, San Jose, Calif.).

Mini-Hepcidin Treatment Regimen

PR65 (FIG. 10A) was selected for the prevention and treatment studies inhepcidin-null mice based on pilot studies in wild-type C57BL/6 mice.PR65 was found to be among the most potent mini-hepcidins and its molarbioactivity after intraperitoneal injection was comparable to that ofnative hepcidin (FIG. 10B). Moreover, PR65 retained full activity withsubcutaneous as compared to intraperitoneal administration (FIG. 10C)and its cost of synthesis was favorable compared to othermini-hepcidins. Based on qualitative assessment of more than 80mini-hepcidins, the high bioactivity of PR65 compared to theprototypical peptide containing the 9 N-terminal amino acids of humanhepcidin (SEQ ID NO:9) is likely due to increased aromaticity,solubility, resistance to proteolysis, as well as lower renal clearancedue to increased plasma protein binding mediated by the palmitoyl group.

To establish optimal dosing parameters for a long-term mini-hepcidintreatment regimen, dose-response (FIG. 11A) and time course (FIG. 11B)experiments in iron-loaded hepcidin knockout mice, Hamp1−/− mice, wasperformed. After 24 hours, subcutaneous injection of 20 and 50 nmoles ofPR65 caused 15% and 10% (p=0.005, p=0.004) decreases in serum iron,while 100 and 200 nmole doses resulted in an 85% and 95% reduction(p<0.001 for both). Because 100 nmoles of PR65 produced a near maximalhypoferremia, this dose was selected for a time course experiment todetermine the timing and duration of its peak effect. The maximal drop(88%) in serum Fe occurred 12 hours after subcutaneous injection(p<0.001), and serum Fe remained severely suppressed (82%) at 24 hoursbut returned to solvent control levels 48 hours after injection.

The activity of PR65 (100 nmoles) was also assessed over 48 hoursthrough its effect on tissue iron retention. Interestingly, spleen ironaccumulation was not observed during 48 hours after PR65 injection (FIG.12). This is likely because the spleen in hepcidin knockout mice iscompletely depleted of iron and it takes more than 2 days to accumulateenough iron so it is conclusively detectable by enhanced Perls stain.Liver iron content, which was already high in these mice, did notvisibly change through the course of the experiment. From 1-4 hoursafter injection, duodenal sections showed distinct iron staining aroundvillous capillary networks indicating continued high ferroportinactivity and uncurbed iron transfer to plasma. From 12-24 hours afterPR65 injection, iron accumulated within enterocytes consistent with theexpected mini-hepcidin-induced loss of ferroportin and diminished irontransfer to plasma. As the mini-hepcidin effect wore off 48 hours afterinjection, iron was no longer retained in enterocytes.

Thus, in some embodiments, subjects are treated with a given does, e.g.about 100 μg/kg, of a mini-hepcidin daily, and after about one week, thedose is halved if the subject's serum iron concentration is below about10 μM or doubled if serum iron is above about 30 μM. At about thebeginning of the third week of treatment, the dose may be increased ordecreased by about 25-50% to maintain serum iron levels between about10-30 μM. In some embodiments, after about 1 week of administration ofone or more mini-hepcidins, the iron levels, and/or ferroportin, and/ormini-hepcidin levels in the subject may be monitored using methods knownin the art or as disclosed herein, and then based on the levels, thesubject may be treated accordingly, e.g. administered one or moresubsequent doses of one or more mini-hepcidins which may be higher orlower than the initial dose. The mini-hepcidins of the subsequent dosesmay be the same or different from the mini-hepcidins of the first dose.

Chronic Administration of Mini-Hepcidin Prevents Iron Loading inHepcidin Deficient Subjects

The ability of PR65 to prevent iron loading in hepcidin in subjects wasexamined using mice as models. Hepcidin KO mice were placed on aniron-deficient diet for 8 weeks to lower their iron stores to a levelcomparable to that of WT mice. After iron depletion, a group of mice wasanalyzed to establish the baseline iron and hematological parameters andthe rest of the mice were placed on an iron-loading diet (300 ppm Fe)for 2 weeks while simultaneously receiving daily subcutaneous injectionsof solven only (control) or PR65 (20, 50 or 100 nmoles) in solvent. Itwas hypothesized that in comparison to the solvent treatment, PR65 wouldcause iron retention in the spleen, decrease serum iron and preventliver iron loading. Because cardiac iron overload is a marker for poorprognosis in iron-loaded patients, heart iron was also measured.Hemoglobin concentrations were monitored to detect potentialiron-restrictive effects of hepcidin excess on erythropoiesis.

Hepcidin agonist activity of the mini-hepcidins was confirmed in alltreated groups by the increased retention of iron in macrophagesmanifested as increases in spleen iron content. Compared to the almostundetectable non-heme iron content in solvent-injected control spleens,all three mini-hepcidin doses caused 15- to 30-fold increases in spleeniron content (p=0.01 for all) (FIG. 13A). Serum iron did not change inmice that received 20 nmoles of PR65 daily (p=0.26), but decreased by69% and 83% in mice that received 50 and 100 nmoles per day (p=0.01 forboth) (FIG. 13B). The decrease in circulating iron concentrations wasalso reflected as dose-dependent 3 and 5 g/dL reductions of hemoglobinconcentrations in the 50 and 100 nmoles groups, respectively (p<0.001for both), but hemoglobin levels did not change significantly at 20nmoles (p=0.13) (FIG. 13C). Heart iron concentration dropped 33%, 60%and 47% in mice treated with 20, 50 and 100 nmoles of PR65 respectively(p=0.08, 0.007, 0.05) (FIG. 13D). Additionally, mice treated with thethree PR65 doses had 76%, 53% and 68% less liver iron thansolvent-treated controls (p=0.001, 0.06, 0.01) and no statisticallysignificant increases in liver iron compared to mice from theiron-depleted baseline group. Except for serum iron and hemoglobin, thelack of a consistent dose-response relationship may indicate that themaximum effect was reached at a relatively low dose so that thedifferences reflect statistical fluctuations (liver and heart iron) orthat two or more effects of PR65 interact in a complex manner (spleniciron may reflect the combined effects of decreased iron export from thespleen, decreased iron absorption in the duodenum, and decreased numberof erythrocytes all of which are expected effects of PR65).

Perls stains of organ sections from mini-hepcidin-treated mice comparedto iron-depleted baseline mice indicated that iron stores in the liverdid not increase from baseline in the 20 and 50 nmoles groups, and wereeven lower than baseline at the 100 nmole dose (FIG. 14). In contrast,liver sections of the solvent-injected mice showed very high ironlevels. A similar pattern of differences between the solvent and PR65groups was observed in the heart, with a complete lack of iron stainingin the heart of mice that received 100 nmoles of peptide. Significantaccumulation of iron in the red pulp of the spleen was observed in allmini-hepcidin groups, but not in mice that received solvent, or inbaseline iron-depleted mice. Duodenal sections at baseline showed noiron staining, while PR65-treated mice showed iron retention in theduodenal enterocytes, again confirming that PR65 blocked iron effluxfrom enterocytes.

Thus, in some embodiments, the present invention provides methods ofpreventing iron loading in subjects having abnormally low or no levelsof hepcidin which comprises chronic administration of one or moremini-hepcidins according to the present invention.

Mini-Hepcidin Effect in Iron Overloaded Hepcidin Knockout Mice

To assess the potential of mini-hepcidins as a standalone treatment foriron overload in subjects, 12 week old iron-overloaded hepcidin knockoutmice were injected with 50 nmoles of PR65 daily for 14 days. This dosewas chosen as the maximal tolerated dose because mice that received 100nmoles in the previous experiment became moderately anemic. Peptideactivity was confirmed by the 15-fold increase in spleen iron content(p<0.001) (FIG. 15A). In contrast to mice that were iron-depleted beforePR65 administration, in mice with established iron overload serum ironlevels were not decreased 24 hours after the last dose compared tosolvent-treated mice (p=0.682) (FIG. 15B). However, the 2 g/dL decreasein hemoglobin (p=0.012) (FIG. 15C) suggests that serum iron could havebeen transiently decreased during the treatment. The less than 24 hoursduration of the hypoferremic effect of each 50 nmoles dose may have beendue to the severe iron overload of the hepcidin knockout mice at thisage. Accumulated hepatocyte iron could stimulate ferroportin synthesisand iron efflux from hepatocytes into plasma by relieving the inhibitionof ferroportin translation by iron-regulatory proteins (IRPs)interacting with 5′ iron-regulatory element (5′ IRE) in the ferroportinmRNA, and possibly by other mechanisms. Compared to the control group,iron-loaded mice treated with PR65 showed a trend toward decreased heartiron concentrations (24% decrease, p=0.085) (FIG. 15D), and their liveriron levels decreased by about 20% (p=0.009) or about 200 μg/g (FIG.15E), an amount equivalent to the total hepatic iron content inwild-type mice.

Enhanced Perls staining demonstrated iron retention in the spleen andduodenum in mice treated with PR65 and a change in the iron distributionpattern in the liver (FIG. 16). No statistically significant differenceswere noticeable in the heart and pancreas sections of the solvent andPR65-treated mice. In the aggregate, staining and quantitative analysisindicate that the 2-week mini-hepcidin treatment alone could not onlyinhibit dietary iron absorption but also redistribute a modest amount ofiron from the liver to the spleen.

Thus, in some embodiments, human subjects being treated for an ironoverload disease are treated with one or more mini-hepcidins for aperiod of at least the minimum period necessary to detect that thetreatment prevented liver iron accumulation by available imagingtechnologies (e.g. FerriScan), e.g. about three months. In somesubjects, the treatment may be continued for many years or for the lifeof the subject.

As shown in FIG. 13, PR65 acted predominantly by reducing ironabsorption but also redistributed iron into splenic macrophages whenadministered prophylactically. Thus, in some embodiments, one or moremini-hepcidins may be administered to a subject as a prophylactictreatment against iron overload in the subject. For example, where asubject having liver iron that is within a normal range, but has apredisposition for an iron overload disease (e.g. geneticallypredisposed to excessive iron adsorption), or is at risk of developingan abnormally high level of iron, the subject may be administered one ormore mini-hepcidins to prevent and/or reduce the likelihood that thesubject will develop an iron overload disease and/or abnormally highlevels of iron.

According to FIG. 13, iron distribution was calculated based on thefollowing assumptions and equations: Estimated organ masses based onaverage weight of 25 g, Blood volume (5.5%)=1.4 ml, Liver mass (5%)=1.3g, Spleen mass (0.3%)=0.08 g, % Fe in Hb=0.34% based on the molecularmass of Hb=64,000 Dalton and 4 iron atoms with a total atomic mass of224 Dalton, Total iron in Hb=(Hb concentration)×(Blood volume)×(% Fe inHb), and Total iron in an organ=molar iron concentration×organ mass×56g/mole. The amounts for the solvent group and the PR65 group are shownas follows:

TABLE 5 Total organ iron (mg) Solvent treatment Hb = 15 g/dl 0.7 Liveriron concentration = 17 μmoles/g 1.2 Spleen iron concentration = 0.5μmoles/g 0.002 Total 1.9 PR65: 50 nmoles Hemoglobin = 11.5 g/dl 0.5Liver iron concentration = 8 μmoles/g 0.6 Spleen iron concentration = 30μmoles/g 0.1 Total 1.2 (36% decrease)

The resulting decrease of plasma iron could also reduce the levels oftoxic non-transferrin bound iron (NTBI) and promote the mobilization ofiron from the heart and endocrine organs where iron excess is nottolerated. Thus, in some embodiments, one or more mini-hepcidins may beadministered to a subject in order to reduce the levels of NTBI and/orpromote the mobilization of iron from the heart and endocrine organs toother organs and tissues.

Unlike phlebotomy or chelation, mini-hepcidins would not be expected toappreciably increase iron losses from the body. In a relatively mildmodel of iron overload in HFE null mice (Viatte L, et al. (2006) Blood107(7):2952-2958), transgenic hepcidin expression was reported to causesignificant redistribution of iron into hepatic macrophages, a locationwhere iron accumulation is relatively nontoxic. In more overloadedHamp1^(−/−) mice, red pulp macrophages in mini-hepcidin-treated miceretained iron but the small resulting decrease in liver and heart ironstores suggests that mini-hepcidins alone confer a modest therapeuticbenefit once the liver iron burden is high. The shorter than 24 houreffect of a mini-hepcidin dose on transferrin saturation in this severeiron overload model may imply that NTBI continues to deliver iron toparenchymal organs counteracting the effects of iron redistribution tomacrophages and decreased iron absorption.

Therefore, in established iron overload in human subjects, effectivetreatment with one or more mini-hepcidins may include more than one doseper day, a prolonged treatment period before a beneficial effect inliver iron can be detected, or may be combined with removal of iron byphlebotomy or chelation.

Dosages

The exclusive use of L-amino acids in PR65 was found to significantlyreduce peptide production costs. In addition, the unnatural and highlyaromatic residues in PR65 were unexpectedly found to substantiallyreduce the minimal effective dose in mice to 20 nmoles/d or 1.3 mg/kg/d.

According to U.S. Food and Drug Administration dosing adjustmentguidelines, the difference in metabolic rates between the mouse andhuman requires a conversion based on the Km factor which normalizesdoses to body surface area (Reagan-Shaw S, et al. (2008) FASEB J22(3):659-661). A human equivalent dose (HED) can be estimated byHED=animal dose (mg/kg)×(animal Km/human Km), where the Km for mouse andan adult human are 3 and 37, respectively. Thus, according to thepresent invention, a subcutaneous dose of mini-hepcidin in a human couldbe up to about 50-100 μg/kg/d, about 75-125 μg/kg/d, or about 90-110μg/kg/d, preferably about 100 μg/kg/d (as this dose is a readilyadministrable amount of peptide about three times the median basal doseof the most widely used peptide drug, subcutaneous insulin, commonlyused at 0.75 U/kg/d or 33 μg/kg/d in type 2 diabetics (Rosenstock J, etal. (2001) Diabetes Care 24(4):631-636)). Of course, lower doses, aswell as higher doses, depending on the particular mini-hepcidin, form ofadministration, formulation, the subject and the degree of ironoverload, may be administered to subject.

Important differences between murine and human iron metabolism thatcould alter the effect of a mini-hepcidin, e.g. PR65, in humans includethe somewhat longer lifetime of human erythrocytes (120 days vs 40 days)and the much lower fractional iron losses in humans (daily iron lossescompared to total body iron) as estimated from the slower depletion ofiron stores on iron-deficient diets (in males: 300-600 days vs 15-20days). The net effect of these differences is the much lowercontribution of intestinal iron absorption to the daily iron flux inhumans (4-8% compared to more than 50% in mice) (Ramos E, et al. (2011)Hepatology 53(4):1333-1341). If hepcidin and its analogs exert strongereffects on macrophages than on enterocytes (Reagan-Shaw S, et al. (2008)FASEB J 22(3):659-661) this could further decrease the relative doses ofmini-hepcidins required for a similar hypoferremic effect in humans.Thus, in some embodiments, a therapeutically effective dose of one ormore mini-hepcidins ranges from about 10-500 μg/kg/d. Again, lowerdoses, as well as higher doses, depending on the particularmini-hepcidin, form of administration, formulation, the subject and thedegree of iron overload, may be administered to subject.

As provided herein, mini-hepcidins according to the present inventionmay be used to inhibit, reduce, or treat iron overload in subjects atrisk due to genetic defects or those who have already undergone irondepletion, but no longer tolerate chelation or venesection therapy. Themini-hepcidins according to the present invention may be used to treat asubject having β-thalassemia major and/or a subject having hepcidinlevels that are higher than normal but are lower than what isappropriate for the degree of iron overload and the particular subject.For example, one or more min-hepcidins according to the presentinvention may be used to treat a subject who suffers fromhyperabsorption of dietary iron, but has normal levels of iron, in orderto lower the amount of iron in the subject and offset thehyperabsorption. One or more mini-hepcidins according to the presentinvention may be used to treat ineffective erythropoiesis and improveanemia in subjects.

Because of the relatively small size of the mini-hepcidins of thepresent invention, the mini-hepcidins may be appropriately formulatedand optimized for oral administration or administration by othernoninvasive means such as those used for insulin administration (RoachP. (2008) Clinical Pharmacokinetics 47(9):595-610) such as inhalation,or transcutaneous delivery, or mucosal nasal or buccal delivery.

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations, andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

1. An isolated peptide having the following structural formula IA or IBA1-A2-A3-A4-A5-A6-A7-A8-A9-A10  IAA10-A9-A8-A7-A6-A5-A4-A3-A2-A1  IB wherein A1 is Asp, D-Asp, Glu, D-Glu,pyroglutamate, D-pyroglutamate, Gln, D-Gln, Asn, D-Asn, bhAsp, Ida,Ida(NHPal), or N-MeAsp; A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile,D-Ile, Ala, D-Ala, Tle, Inp, Chg, bhThr, or N-MeThr; A3 is His, D-His,Asn, D-Asn, Arg, D-Arg, L-His(π-Me), D-His(π-Me), L-His(τ-Me), orD-His(τ-Me); A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr,D-Tyr, Phg, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, Amc, PheF5, hPhe, Igl,or cyclohexylalanine; A5 is Pro, D-Pro, Ser, D-Ser, Oic, bhPro,trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, or Idc; A6 is Arg, D-Arg, Thr,D-Thr, Lys, D-Lys, D Nω,ω-dimethyl-arginine, L-Nω,ω-dimethyl-arginine,D-homoarginine, L-homoarginine, D-norarginine, L-norarginine,citrulline, a modified Arg wherein the guanidinium group is modified orsubstituted, or N-MeArg; A7 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala,Cys(S-tBut), homoCys, Pen, (D)Pen, S-tertiary butyl-cysteine,Cys(S-S-Pal), Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), orCys(S-S-Cys); A8 is Arg, D-Arg, D-Ile, Leu, D-Leu, Thr, D-Thr, Lys,D-Lys, Val, D-Val, D-Nω,ω-dimethyl-arginine, L-Nω,ω-dimethyl-arginine,D-homoarginine, L-homoarginine, D-norarginine, L-norarginine,citrulline, a modified Arg wherein the guanidinium group is modified orsubstituted, Norleucine, norvaline, bhIle, Ach, N-MeArg, or N-MeIle; A9is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Tyr, D-Tyr, Trp, D-Trp,Phe-R^(a), D-Phe-R^(a), Dpa-R^(a), D-Dpa-R^(a), Trp-R^(a), bhPhe-R^(a),PheF5, N-MePhe, benzylamide, 2-aminoindane, bhPhe, Dpa, Bip, 1Nal, 2Nal,bhDpa, or cyclohexylalanine, which may or may not have R^(a) linkedthereto, wherein R^(a) is palmitoyl-PEG wherein PEG is PEG11 orminiPEG3, palmitoyl-PEG-PEG wherein PEG is PEG11 or miniPEG3, butanoyl(C4)-PEG11-, octanoyl (C8, Caprylic)-PEG11-, palmitoyl (C16)-PEG11-, ortetracosanoyl (C24, Lignoceric)-PEG11-; and A10 is Cys, D-Cys, Ser,D-Ser, Ala, D-Ala, Ida, Ida(NHPal)Ahx, or Ida(NBzl2)Ahx; wherein thecarboxy-terminal amino acid is in amide or carboxy-form; wherein atleast one sulfhydryl amino acid is present as one of the amino acids inthe sequence; and wherein A1, A1 to A2, A10, or a combination thereofare optionally absent, with the proviso that the peptide is not one ofthe peptides as set forth in Table
 1. 2. The peptide according to claim1, wherein the peptide contains at least one of the following: a)A1=N-MeAsp, Ida, or Ida(NHPal); b) A5=bhPro; c) A6=D-Val, D-Leu, Lys,D-Lys, Arg, D-Arg, Ach, bhArg, or N-MeArg; d) A7=Cys(S-S-Pal),Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys); and/or e)A8=D-Val, D-Leu, Lys, D-Lys, Arg, D-Arg, Ach, bhArg, or N-MeArg.
 3. Thepeptide according to claim 1, wherein: i) when A1 is Ida and A9 is Phe,then A10 is not Ahx-Ida(NHPal); ii) when A1 is Ida, A9 is notbhPhe-R^(b), wherein R^(b) is S-(palmityl)thioglycolic-PEG-; iii) whenA4 is D-Phe, A7 is not D-Cys(S-S-tBut) and A9 is not D-Trp-R^(c),wherein R^(c) is Butanoyl-PEG11-, Octanoyl-PEG11-, Palmitoyl-PEG11-, orTetracosanoyl-PEG11-; and iv) when A1 is Ida and A9 is bhPhe-R^(d),wherein R^(d) is palmitoyl-PEG-miniPEG3-, A6 and A8 are not both D-Argor both bhArg.
 4. The peptide according to claim 1, wherein A1 is D-Asp,N-MeAsp, Ida, or Ida(NHPal); A2 is Thr or D-Thr; A3 is His or D-His; A4is Dpa or D-Dpa; A5 is Pro, D-Pro, bhPro, or Oic; A6 is Ile, D-Ile, Arg,D-Val, D-Leu, Ach, or N-MeArg; A7 is Cys, D-Cys, Cys(S-S-Pal),Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys); A8 is Ile,D-Ile, Arg, D-Val, D-Leu, Ach, or N-MeArg; A9 is Phe, D-Phe, Dpa, D-Dpa,Trp, D-Trp, bhPhe, Phe-R^(a), D-Phe-R^(a), Dpa-R^(a), D-Dpa-R^(a),Trp-R^(a), bhPhe-R^(a), wherein R^(a) is palmitoyl-PEG wherein PEG isPEG11 or miniPEG3, palmitoyl-PEG-PEG wherein PEG is PEG11 or miniPEG3,butanoyl (C4)-PEG11-, octanoyl (C8, Caprylic)-PEG11-, palmitoyl(C16)-PEG11-, or tetracosanoyl (C24, Lignoceric)-PEG11-; and A10, ifpresent, is Ida(NHPal)Ahx or Ida(NBzl2)Ahx.
 5. The peptide of accordingto claim 1, wherein the peptide is selected from the group consistingof: PR42′, PR47, PR48, PR49, PR50, PR51, PR52, PR53, PR56, PR57, PR58,PR59, PR60, PR61, PR63, PR65, PR66, PR67, PR68, PR69, PR70, PR71, PR72,PR74, and PR82.
 6. The peptide according to claim 1, wherein the peptideexhibits hepcidin activity.
 7. The peptide according to claim 1, whereinthe peptide binds ferroportin.
 8. A composition which comprises at leastone peptide according to claim
 1. 9. A method of binding a ferroportinor inducing ferroportin internalization and degradation which comprisescontacting the ferroportin with at least one peptide according to claim1 or a composition comprising the at least one peptide.
 10. A method oftreating a disease of iron metabolism in a subject which comprisesadministering at least one peptide according to claim 1 or a compositioncomprising the at least one peptide to the subject.
 11. The method ofclaim 10, wherein the disease of iron metabolism is an iron overloaddisease.
 12. A kit comprising at least one peptide according to claim 1or a composition comprising the at least one peptide packaged togetherwith a reagent, a device, instructional material, or a combinationthereof.
 13. A complex comprising at least one peptide according toclaim 1 bound to a ferroportin or an antibody. 14-15. (canceled)
 16. Themethod according to claim 10, wherein the at least one peptide isadministered at an effective daily dose as a single daily dose or asdivided daily doses.
 17. The method according to claim 16, wherein theeffective daily dose is about 10-500 μg/kg/day and the at least onepeptide is provided in a formulation for subcutaneous injection.
 18. Themethod according to claim 16, wherein the effective daily dose is about10-1000 μg/kg/day and the at least one peptide is provided in aformulation for oral, pulmonary, parenteral, or mucosal administration.